fluorescent dyes attached. Alternatively, proteins can be labeled with fluorescent
dyes for MST, and an aptamer can be used as the non-fluorescent, titrated interaction
partner.
2.1.5 Isothermal Titration Calorimetry (ITC) [2, 8]
The calorimetric method ITC directly measures the heat released or consumed in
the course of a molecular binding event. The technology offers high information
content. Besides thermodynamic parameters such as ΔH, ΔS, and ΔG, the
equilibrium-binding affinity (K D ) and interaction stoichiometry can be determined
from the same experiment. Recent developments even allow to study kinetics by ITC
[18]. Hence ITC offers the highest information content.
In a typical ITC experiment, one interaction partner is put into a reaction cell at a
constant volume and concentration, whereas the other partner is titrated into the
reaction cell via a rotating syringe. ITC allows to study aptamer-target interactions
without modification of the molecules.
2.1.6 Fluorescence Polarization or Fluorescence Anisotropy (FP or FA)
[9, 10]
FP (often called FA) is based on the phenomenon that the polarization plane of
emitted light of a small fluorescent molecule (excited with plane-polarized light)
changes upon binding of an interaction partner. FP enables to calculate the affinity
(K D ) of the aptamer-target interaction.
In a typical FP or FA experiment, one binding partner (the smaller one) is
monitored via an attached fluorescent dye and held at constant concentration during
the experiment, whereas the other binding partner is titrated across a certain concentration range.
2.1.7 Flow Cytometry [11, 12]
This optical method is commonly used to quantify the interaction strength between
aptamers and whole cells, by sorting populations of cells that show interaction to
fluorescently labeled aptamers, combined by quantification of the fluorescence
signal coming from the aptamers. The steady-state affinity (K D ) can be derived
from flow cytometry assays, in which the target cells are incubated with increasing
concentrations of fluorescently labeled aptamers.
In a typical flow cytometry experiment, cells are incubated with fluorescent
aptamers.
Biophysical Characterization of Aptamer-Target Interactions
7
dyes for MST, and an aptamer can be used as the non-fluorescent, titrated interaction
partner.
2.1.5 Isothermal Titration Calorimetry (ITC) [2, 8]
The calorimetric method ITC directly measures the heat released or consumed in
the course of a molecular binding event. The technology offers high information
content. Besides thermodynamic parameters such as ΔH, ΔS, and ΔG, the
equilibrium-binding affinity (K D ) and interaction stoichiometry can be determined
from the same experiment. Recent developments even allow to study kinetics by ITC
[18]. Hence ITC offers the highest information content.
In a typical ITC experiment, one interaction partner is put into a reaction cell at a
constant volume and concentration, whereas the other partner is titrated into the
reaction cell via a rotating syringe. ITC allows to study aptamer-target interactions
without modification of the molecules.
2.1.6 Fluorescence Polarization or Fluorescence Anisotropy (FP or FA)
[9, 10]
FP (often called FA) is based on the phenomenon that the polarization plane of
emitted light of a small fluorescent molecule (excited with plane-polarized light)
changes upon binding of an interaction partner. FP enables to calculate the affinity
(K D ) of the aptamer-target interaction.
In a typical FP or FA experiment, one binding partner (the smaller one) is
monitored via an attached fluorescent dye and held at constant concentration during
the experiment, whereas the other binding partner is titrated across a certain concentration range.
2.1.7 Flow Cytometry [11, 12]
This optical method is commonly used to quantify the interaction strength between
aptamers and whole cells, by sorting populations of cells that show interaction to
fluorescently labeled aptamers, combined by quantification of the fluorescence
signal coming from the aptamers. The steady-state affinity (K D ) can be derived
from flow cytometry assays, in which the target cells are incubated with increasing
concentrations of fluorescently labeled aptamers.
In a typical flow cytometry experiment, cells are incubated with fluorescent
aptamers.
Biophysical Characterization of Aptamer-Target Interactions
7