example, due to binding of a ligand, leads to different interference patterns at this
reflective layer. This, in turn, causes a shift of the interference spectrum to different
wavelengths. From the time-resolved monitoring of this shift, it is possible to derive
real-time association (k on ) and dissociation rates (k off ) of an aptamer-target interaction. The steady-state affinity (K D ) can be extracted from equilibrium titrations. As
for SPR analyses, repeat of BLI experiments at different temperatures allows for
determination of thermodynamic parameters.
In a typical BLI experiment, one of the interaction partners is immobilized to the
sensor tip, whereas the other partner is supplied in different concentrations in a
microwell plate. As for SPR, numerous coupling methods exist that allow to analyze
aptamers both as the immobilized and the in-solution interaction partner.
2.1.3 SwitchSENSE (SwS) [16]
The SwitchSENSE technology monitors voltage-driven movement of DNA nanolevers attached to a sensor surface. Usually, such a nano-lever carries one of the
interaction partners by direct, covalent attachment. Binding of the other partner
affects the hydrodynamic friction of the nano-lever and hence its movement on the
sensor surface, which can be monitored through time-resolved single-photon
counting. Kinetic parameters (k on and k off ) and steady-state affinity (K D ) can be
extracted. Furthermore, thermodynamic parameters can be estimated by analyses at
different temperatures.
In a typical SwitchSENSE experiment, one interaction partner is immobilized to
the nano-levers on the sensor surface, whereas the other interaction partner is titrated
in different concentrations. Aptamers can often be coupled directly to the nucleic
acid-based nano-levers by base-pairing.
2.1.4 MicroScale Thermophoresis (MST) [3, 13, 17]
The optical method MST is based on the combined effect of Temperature-Related
Intensity Change of fluorescent molecules (TRIC) and their directed movement
along temperature gradients (thermophoresis). Both the TRIC effect and the thermophoretic component of the MST signal vary with three key molecular features that
change upon binding between an aptamer and its target: molecular size, molecular
charge, as well as the hydration shell of the molecules. Information on steady-state
binding affinity (K D ) can be directly obtained from a ligand titration. By variation of
assay temperatures, also thermodynamics can be determined.
In a typical MST experiment, one binding partner is held at a constant concentration and is monitored for its TRIC effect and thermophoretic movement by its
intrinsic fluorescence or by a coupled fluorescent dye. The other binding partner is
titrated usually in 16 dilution steps in order to sample a very large ligand concentration range. Aptamers can be used in MST very straightforward as the constant,
fluorescent interaction partner, because they can be easily obtained with all kinds of
6
M. Plach and T. Schubert
reflective layer. This, in turn, causes a shift of the interference spectrum to different
wavelengths. From the time-resolved monitoring of this shift, it is possible to derive
real-time association (k on ) and dissociation rates (k off ) of an aptamer-target interaction. The steady-state affinity (K D ) can be extracted from equilibrium titrations. As
for SPR analyses, repeat of BLI experiments at different temperatures allows for
determination of thermodynamic parameters.
In a typical BLI experiment, one of the interaction partners is immobilized to the
sensor tip, whereas the other partner is supplied in different concentrations in a
microwell plate. As for SPR, numerous coupling methods exist that allow to analyze
aptamers both as the immobilized and the in-solution interaction partner.
2.1.3 SwitchSENSE (SwS) [16]
The SwitchSENSE technology monitors voltage-driven movement of DNA nanolevers attached to a sensor surface. Usually, such a nano-lever carries one of the
interaction partners by direct, covalent attachment. Binding of the other partner
affects the hydrodynamic friction of the nano-lever and hence its movement on the
sensor surface, which can be monitored through time-resolved single-photon
counting. Kinetic parameters (k on and k off ) and steady-state affinity (K D ) can be
extracted. Furthermore, thermodynamic parameters can be estimated by analyses at
different temperatures.
In a typical SwitchSENSE experiment, one interaction partner is immobilized to
the nano-levers on the sensor surface, whereas the other interaction partner is titrated
in different concentrations. Aptamers can often be coupled directly to the nucleic
acid-based nano-levers by base-pairing.
2.1.4 MicroScale Thermophoresis (MST) [3, 13, 17]
The optical method MST is based on the combined effect of Temperature-Related
Intensity Change of fluorescent molecules (TRIC) and their directed movement
along temperature gradients (thermophoresis). Both the TRIC effect and the thermophoretic component of the MST signal vary with three key molecular features that
change upon binding between an aptamer and its target: molecular size, molecular
charge, as well as the hydration shell of the molecules. Information on steady-state
binding affinity (K D ) can be directly obtained from a ligand titration. By variation of
assay temperatures, also thermodynamics can be determined.
In a typical MST experiment, one binding partner is held at a constant concentration and is monitored for its TRIC effect and thermophoretic movement by its
intrinsic fluorescence or by a coupled fluorescent dye. The other binding partner is
titrated usually in 16 dilution steps in order to sample a very large ligand concentration range. Aptamers can be used in MST very straightforward as the constant,
fluorescent interaction partner, because they can be easily obtained with all kinds of
6
M. Plach and T. Schubert