different aptamers to one target or the specificity of an aptamer to one or few of
multiple targets.
Binding kinetics describes the time-dependent, dynamic component of the binding event between aptamer and target. The association rate constant k a (k on , in
M
À1 s
À1 ) describes the association of aptamer and target to the binary (or higher
order) complex over time. The dissociation rate constant k d (k off , in s
À1
) describes the
rate of dissociation of aptamer and target and is hence a measure of temporal stability
of the aptamer-target complex. In order to ensure proper functionality of an aptamer
in its final application (e.g., in diagnostic use), aptamers with desired binding
kinetics may be chosen during the development phase.
Binding thermodynamics describe the enthalpic (ΔH) and entropic (ÀTΔS)
parameters of the interaction between aptamer and target, which will only occur
spontaneously when the Gibbs free energy (ΔG) of the interaction is negative (either
enthalpy or entropy driven). The enthalpic parameter ΔH is the energy change
resulting from the formation of non-covalent interactions between aptamer and
target and the changes of hydrogen bond and van der Waals interactions between
aptamer, target, and the solvent. The entropic counterpart ΔS represents the global
thermodynamic property of the system, hence the degree of freedom of the system.
Thermodynamic parameters are thus helpful to understand the molecular principles
of aptamer-target interactions and to optimize aptamers with respect to certain
thermodynamic characteristics.
In the following, different biophysical methods to characterize aptamer-target
interactions are described. Besides the seven well-established and broadly applied
methods, Surface Plasmon Resonance (SPR) [1–5], Biolayer Interferometry (BLI)
[6, 7], Isothermal Titration Calorimetry (ITC) [2, 8], Fluorescence Polarization
(FP or FA) [9, 10], Flow Cytometry [11, 12], Filter-Binding Assay (FB)/Filter
Retention Assay [4, 13], and Electromobility Shift Assay (EMSA) [14, 15], two
emerging technologies SwitchSENSE (SwS) [16] and MicroScale Thermophoresis
(MST) [3, 13, 17] are discussed.
2 Biophysical Techniques to Study Basic Binding
Parameters of Aptamers and Their Target Molecules
Numerous physical and biophysical methods and technologies are available today
for determining the aforementioned basic binding parameters of aptamer-target
interactions. The methods differ with respect to the type of physical readout and
their information content. Some determine binding parameters in an indirect manner,
others by direct readout; some work in solution, whereas others require immobilization of either target or aptamer to a solid phase. Modification-free, label-free, or
fluorescent technologies are available. Some methods characterize interactions in a
steady-state equilibrium, whereas others analyze dynamic binding kinetics. Some
techniques are capable of studying interactions between aptamers and whole cells,
Biophysical Characterization of Aptamer-Target Interactions
3
multiple targets.
Binding kinetics describes the time-dependent, dynamic component of the binding event between aptamer and target. The association rate constant k a (k on , in
M
À1 s
À1 ) describes the association of aptamer and target to the binary (or higher
order) complex over time. The dissociation rate constant k d (k off , in s
À1
) describes the
rate of dissociation of aptamer and target and is hence a measure of temporal stability
of the aptamer-target complex. In order to ensure proper functionality of an aptamer
in its final application (e.g., in diagnostic use), aptamers with desired binding
kinetics may be chosen during the development phase.
Binding thermodynamics describe the enthalpic (ΔH) and entropic (ÀTΔS)
parameters of the interaction between aptamer and target, which will only occur
spontaneously when the Gibbs free energy (ΔG) of the interaction is negative (either
enthalpy or entropy driven). The enthalpic parameter ΔH is the energy change
resulting from the formation of non-covalent interactions between aptamer and
target and the changes of hydrogen bond and van der Waals interactions between
aptamer, target, and the solvent. The entropic counterpart ΔS represents the global
thermodynamic property of the system, hence the degree of freedom of the system.
Thermodynamic parameters are thus helpful to understand the molecular principles
of aptamer-target interactions and to optimize aptamers with respect to certain
thermodynamic characteristics.
In the following, different biophysical methods to characterize aptamer-target
interactions are described. Besides the seven well-established and broadly applied
methods, Surface Plasmon Resonance (SPR) [1–5], Biolayer Interferometry (BLI)
[6, 7], Isothermal Titration Calorimetry (ITC) [2, 8], Fluorescence Polarization
(FP or FA) [9, 10], Flow Cytometry [11, 12], Filter-Binding Assay (FB)/Filter
Retention Assay [4, 13], and Electromobility Shift Assay (EMSA) [14, 15], two
emerging technologies SwitchSENSE (SwS) [16] and MicroScale Thermophoresis
(MST) [3, 13, 17] are discussed.
2 Biophysical Techniques to Study Basic Binding
Parameters of Aptamers and Their Target Molecules
Numerous physical and biophysical methods and technologies are available today
for determining the aforementioned basic binding parameters of aptamer-target
interactions. The methods differ with respect to the type of physical readout and
their information content. Some determine binding parameters in an indirect manner,
others by direct readout; some work in solution, whereas others require immobilization of either target or aptamer to a solid phase. Modification-free, label-free, or
fluorescent technologies are available. Some methods characterize interactions in a
steady-state equilibrium, whereas others analyze dynamic binding kinetics. Some
techniques are capable of studying interactions between aptamers and whole cells,
Biophysical Characterization of Aptamer-Target Interactions
3