Chapter 8
switchSENSE Technology for Analysis of DNA Polymerase
Kinetics
Guillaume Bec and Eric Ennifar
Abstract
The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of
interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at
one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE
approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics,
dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to
some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that
allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate
of the enzyme.
Key words HIV reverse transcriptase, switchSENSE technology, Kinetics, Biosensor, DNA
polymerase
1 Introduction
Immobilization of molecules on solid supports is a fairly wellknown method that allows for very sensitive measurement of
molecular interaction, as for the very popular surface plasmon
resonance (SPR) approach [1]. Usually, biophysical properties
such as molecular size or hydrodynamic radius are inferred from
the molecular mobility under the influence of external forces.
switchSENSE technology is a combination of these two approaches
with the measurement of analyte adsorption on a layer of actuated
surface-bound fluorescent probe [2, 3]. Two main measurement
modes are accessible: (1) a static mode, where analyte binding is
measured thanks to fluorescence intensity variation, and (2) a
dynamic mode, where binding is detected through the change of
the oscillation rate of the probe actuated by an AC electric field.
These two alternative measurement modes give access to a wide
range of biophysical parameters as k ON , k OFF , K D , hydrodynamic
diameter, or conformational changes (see for instance refs. 4–11).
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
145
switchSENSE Technology for Analysis of DNA Polymerase
Kinetics
Guillaume Bec and Eric Ennifar
Abstract
The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of
interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at
one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE
approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics,
dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to
some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that
allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate
of the enzyme.
Key words HIV reverse transcriptase, switchSENSE technology, Kinetics, Biosensor, DNA
polymerase
1 Introduction
Immobilization of molecules on solid supports is a fairly wellknown method that allows for very sensitive measurement of
molecular interaction, as for the very popular surface plasmon
resonance (SPR) approach [1]. Usually, biophysical properties
such as molecular size or hydrodynamic radius are inferred from
the molecular mobility under the influence of external forces.
switchSENSE technology is a combination of these two approaches
with the measurement of analyte adsorption on a layer of actuated
surface-bound fluorescent probe [2, 3]. Two main measurement
modes are accessible: (1) a static mode, where analyte binding is
measured thanks to fluorescence intensity variation, and (2) a
dynamic mode, where binding is detected through the change of
the oscillation rate of the probe actuated by an AC electric field.
These two alternative measurement modes give access to a wide
range of biophysical parameters as k ON , k OFF , K D , hydrodynamic
diameter, or conformational changes (see for instance refs. 4–11).
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
145