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European Biopharmaceutical Review
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Access to basic binding parameters is essential for the design and
development of drugs, antibodies, aptamers, and other diagnostic or
therapeutic molecules. Currently, various methods are used to determine
these parameters – for example, binding affinity, stoichiometry,
thermodynamics and kinetics. Assays range from semi-quantitative
approaches – including pull-down assays or electromobility shift assays –
to quantitative methods – such as surface plasmon resonance (SPR),
isothermal titration calorimetry (ITC), fluorescence anisotrophy,
biolayer interferometry and fluorescence correlation spectroscopy.
SPR
is a commonly-used method that measures changes in the index of
refraction at the surface where a binding event occurs between an
immobilised molecule on a thin metal film and its ligand in solution.
This sensitive technology allows for time-resolved measurements of
binding events to the surface-immobilised molecule. It can be used to
establish kinetics, stoichiometry and affinities from sub nM to mM
(dissociation constant KD). Despite drawbacks such as intense laboratory
set-up, necessary modification of one interaction partner by
immobilisation and influence of mass transport limitations on data
quality, SPR is the method of choice if Kon and Koff rates are to be
determined.
Label-free ITC enables direct access to
thermodynamics, binding affinity (nM to sub mM range) and stoichiometry,
by measuring the dissipated and absorbed heat of a binding reaction
between a target molecule and ligand. However, to be able to detect
these changes in heat, larger amounts of sample are necessary. In
addition, the technology is limited in buffer usage, is laboratory
intense, and only interaction with a higher change in enthalpy can be
measured. Nonetheless, as thermodynamics can be directly measured, ITC
is the method of choice when determining these parameters.
Novel Approach
MicroScale
Thermophoresis (MST) is a novel technology that can be used to
characterise molecular interactions and it has enjoyed considerable
attention in scientific literature (1-3). The basic principle behind
this technique is a physical phenomenon called thermophoresis, which
describes the motion of molecules in temperature fields (3,4). The
thermophoretic effect is highly sensitive to the interface between a
solvent and molecule; binding of a ligand to a target molecule will
alter at least one essential parameter – either size, charge or
hydration shell – which determines the thermophoretic movement of a
molecule. Consequently, the movement of the molecule-ligand complex can
be compared against the action of single molecules. Furthermore, MST can
monitor the movement of molecules in temperature gradients, interpret
this movement as binding events, and use this information to access
basic binding parameters.
Based on an optical system, MST
detects the intrinsic fluorescence of tryptophanes within proteins or
the fluorescence signal of a fluorophore attached to one of the
interaction partners (5). The movement of the fluorescent molecules is
monitored in a μm-sized temperature gradient, with respect to increasing
concentrations of the potential binding partner.
With MST, the
optics focus on the centre of thin glass capillaries holding 4-6μl of
sample, in which a microscopic temperature gradient with a diameter of
~50μm and a temperature difference of ΔT of 2-6°C is established by an
infrared (IR) laser. On activation of the laser, thermophoretic
depletion or accumulation of molecules can be observed in the region of
elevated temperature, which is quantified by the Soret coefficient.
Time Trace
Size,
charge and hydration shell determine the thermophoretic behaviour of a
molecule. The binding of a ligand to the target molecule will alter at
least one of these parameters, resulting in changed thermophoretic
movement of the complex, compared to the single molecules. This effect
can be used to study equilibrium constants, such as the dissociation
constant KD. To obtain this information, a serial dilution of the ligand
is first prepared, mixed with a constant concentration of labelled
target molecule, loaded into capillaries, and then analysed in the
instrument by subsequent scanning of each capillary.
Technique Advantages
MST
allows for fast and flexible assay set-up and optimisation, and a KD
value can be obtained within just minutes or hours. MST – as an
in-solution method – offers free choice of buffers, making close to
native measurements in sera or lysates possible (6,7). To obtain
high-quality data, the technique provides integrated quality controls to
detect sticking and aggregation/precipitation in real time. Upon
detection of these effects, technical conditions and buffers can easily
be optimised to ensure an ideal binding environment. Furthermore, as the
thermophoresis of molecules depends on the charge and hydration shell,
there are no limitations in the size of measured interaction partners.
Finally, the dynamic affinity range from pM to mM, together with low
sample consumption, add to the strengths of MST.
MST Applications
Besides
binding affinity, thermodynamics and stoichiometry, MST offers a broad
application range. It can be used for screening assays, competition
assays, and assays with multiple binding partners. In addition, the
technology can gauge the serum stability of interactions. All of these
provide high information content.
MST is able to perform binding
assays in a fluorescently labelled version or in a label-free assay
format. In the fluorescent assay, the optically visible molecule is
detected via the fluorescent dye attached to it. This assay is highly
sensitive, allowing for pM interactions to be detected in crude extracts
or sera. In contrast, the label-free assay uses the intrinsic
fluorescence of tryptophanes within a target protein. This approach
enables the study of protein-target interaction in a label-free manner
without modification of the target protein (5).
Scientists have
used MST to study different interactions between various molecular
classes, such as nucleic acid, protein-nucleic acid, protein-protein,
protein-small molecule and proteincarbohydrate (8-15). Even challenging
binding events like protein-liposome interactions can be investigated
(16,17). Literature also indicates that data derived from MST
measurements have a high overlap with data from ITC and SPR studies,
making this technology perfectly suited for early-phase drug
development, antibody characterisation and aptamer design. Over 600
publications using MST could be found in June 2015.
Innovation Technology
MST
is a novel technique that can be used to characterise the essential
binding parameters of a range of molecular interactions, such as binding
affinity (pM to mM range), stoichiometry and thermodynamics. It is a
fast and flexible approach that allows researchers to study interactions
under close to native conditions – in sera or lysates – while using low
sample material and producing quality controlled data. It has wide
application across drug development, antibody and aptamer
characterisation, epigenetics or basic science.
References
1. Duhr S and Baaske P, GIT Laboratory Journal, 2013
2. Jerabek-Willemsen M et al, Journal of Molecular Structure, 2014
3. Jerabek-Willemsen M et al, Assay Drug Dev Technol 9: p342, 2011
4. Baaske P et al, Angew Chem Int Ed Engl 49: p2,238, 2010
5. Seidel SA et al, Angew Chem Int Ed Engl 51: p10,656, 2012
6. Seidel SA et al, Methods 59: p301, 2013
7. Wienken CJ et al, Nat Commun 1: p100, 2010
8. Zillner K et al, Methods in Molecular Biology 815: p241, 2012
9. Schubert T et al, Mol Cell 48: p434, 2012
10. Pham TH et al, Nucleic Acids Res, 2013
11. Josling GA et al, Cell Host Microbe 17: p741, 2010
12. Xiong X et al, Nature 497: p392, 2013
13. Immekus F et al, ACS Chem Biol 8: p1,163, 2013
14. Patnaik S et al, J Med Chem 55: p5,734, 2012
15. Wong JE et al, Acta Crystallogr D Biol Crystallogr 71: p592, 2015
16. van den Bogaart G et al, J Biol Chem 287: p16,447, 2012
17. van den Bogaart G et al, Nat Struct Mol Biol 18: p805, 2011
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