Enzyme kinetics
Application Note NT-MO-014
On a razor’s edge: watching DNaseI cutting DNA into pieces
Heide Marie Roth1, Moran Jerabek-Willemsen1, Emil Mladenov2, Vladimir Nikolov2, George Iliakis2 and
Stefan Duhr1
1
2
NanoTemper Technologies GmbH, Munich, Germany
Institute of Medical Radiation Biology, University Hospital Essen, Essen, Germany
Abstract
MicroScale Thermophoresis (MST) has been
established to be a powerful technique to
study interactions of molecules in equilibrium
state and to determine dissociation constants.
In this application note, we present that
employing MST we could monitor DNase I
endonuclease activity in real time. We chose
two different approaches to investigate
nuclease activity: in the first approach we
directly measured nuclease activity in the
same capillary in time steps of 3 s. In the
second approach we stopped the reaction
chemically after different incubation periods
and then performed MST measurements. In
both experiments we detect a time dependent
change in thermophoresis we can assign to
DNase I endonuclease activity. Thus, we could
demonstrate that MST is also a valid method to
visualize enzyme kinetics.
Figure 1: Crystal structure of DNase I in complex with DNA
(PDB: 2DNJ)
DNase I is a robust enzyme with Mg2+-dependent
endonuclease activity.
Introduction
MicroScale Thermophoresis (MST) has been
established to be a powerful technique to study
interactions of molecules in equilibrium state and
thus to determine dissociation constants of a
plethora of different sorts of molecules [JerabekWillemsen et al., 2011]. MST does not require
immobilization of molecules and is even sensitive
to binding events just re-arranging the hydration
shell, but lacking a huge difference in size or
charge. Therefore, MST is a very versatile tool to
investigate interactions of biomolecules which are
not easy accessible by any other technique [Seidel
et al., 2013]. In addition to dissociation constants
we seek to also study slow enzyme kinetics using
MST.
In this report we demonstrate that employing MST
we could monitor DNase I endonuclease activity in
real time.
Figure 2: Schematic representation of DNase I nuclease
activity. DNase I is illustrated in blue, Mg2+ is depicted in
yellow, the green boxes represent DNA and DNA fragments
resulting from DNase I nuclease activity. EDTA, shown in
purple, chelates Mg2+ thus inhibiting DNase I nuclease activity.
This enzyme nonspecifically cleaves DNA to
release di-, tri- and oligonucleotide products with
5´-phosphorylated and 3´-hydroxylated ends
[Kunitz, 1950; Vanecko and Iaskowski, 1961].
Moreover, DNase I acts on single- and double1
stranded DNA, chromatin and RNA:DNA hybrids.
Thus, this enzyme is often used as a tool in
molecular biology.
To investigate the nuclease activity of DNase I, we
chose two different approaches: in the first
approach nuclease activity was measured directly
in time steps of 3 s in the same capillary. In the
second approach, the reaction was stopped
chemically after 16 different incubation periods.
The samples were then loaded into capillaries and
subjected to MST measurements. Changes in
thermophoresis over time, which can be attributed
to DNase I endonuclease activity, could be
observed in both experimental set ups. Analyzing
the changes in thermophoresis, we are able to
visualize slow enzyme kinetics.
Results
1. Real time nuclease assay
To study DNase I nuclease activity by MST in a
real time approach, two reaction setups were
prepared.
A
be chelated by EDTA, this sample served as a
negative control (Figure 2). Therefore DNase I is
able to bind, but not to incise the DNA substrate.
Both capillaries were scanned and MST
measurements were performed in an alternating
manner. Each capillary was measured for 3 s: 1 s
laser off, 1 s laser on, 1 s laser off (Figure 3).
Because of the alternating measurement of both
capillaries, this time frame was in total 8 s for each
capillary. However, measuring the negative control
concomitantly to the active DNase I sample, we
can
exclude
potential
artifacts
due
to
photobleaching of the fluorescent dye.
In our measurements we observe that
thermophoresis of the DNA substrate is constant
over time if EDTA is added to the sample. If
however DNase I is not inhibited by depletion of its
cofactor Mg2+ but is present in its active form, we
see a dramatic change of thermophoresis over
time. This change can therefore be assigned to
DNase I nuclease activity and the digestion of the
DNA substrate. The DNase I activity can be
divided into two phases: in the first phase, the
thermophoresis changes in a linear fashion,
whereas in the second phase, the activity reaches
a plateau after around 800 s. Plotting the change
of thermophoresis against time and fitting the data
we can determine a time constant of tau = 247 s.
2. Quenched nuclease assay
B
Figure 3: DNase I endonuclease activity monitored by MST
A. Time traces of DNase I activity were recorded with
1 s/1 s/1 s for IR Laser off/on/off. Data within blue and red
cursors were set to determine the change in concentration due
to thermophoresis. The different thermophoretic motion of full
length compared to incised DNA could then be further
analyzed.
B. Normalized, relative fluorescence change was plotted
against time. The black squares correspond to the sample
comprising active DNase I, whereas the gray squares
correspond to the sample containing DNase I and EDTA
(negative control). Data were fit (red line) and the time constant
was determined to 247 s (± 4 s).
One setup contained buffer with Mg2+, DNase I
and the DNA substrate, the second one
additionally contained EDTA. DNase I requires
Mg2+
as
a
cofactor
to
cleave
the
phosphodiesterbond. Since divalent cations can
DNase I nuclease activity was
assayed by a quenching reaction.
additionally
Figure 4: DNase I endonuclease activity using a quenching
approach. Normalized, relative fluorescence change averaged
of three technical replicates (including standard error) was
plotted against time. This result could be verified by two
independent experiments.
This approach allows us to stop the reaction after
different defined incubation periods. Moreover, the
incubation periods can be chosen independent
from the Monolith instruments settings and thus
could be even less than 3 s. Since DNase I could
be inhibited by EDTA and importantly EDTA does
not significantly influence the fluorescent label of
2
the DNA, EDTA thus is an appropriate tool to stop
DNase I nuclease activity after different incubation
periods to be then assayed with MST.
Using the quenching assay, we again observe a
time dependent change in thermophoresis which
can be assigned to DNase I nuclease activity
(Figure 4). Both types of DNase I nuclease assays
yield very similar and comparable results
indicating that the nuclease activity is not
influenced by the technique employed.
Instrumentation
The measurements were carried out on a
NanoTemper Monolith NT.115 instrument.
The real time measurement was performed at
50 % LED (blue channel) and 20 % MST power,
Laser-On time was 1 s, Laser-Off time 1 s.
The quenching measurement was performed at
50 % LED (blue channel) and 20% MST power,
Laser-On time was 30 s, Laser-Off time 5 s.
Conclusion
References
The study presented here provides the first
example that MicroScale Thermophoresis is also
applicable to measure nuclease activity in real
time. In future, a quenching approach in
combination with a quench-flow device would
allow us to also measure kinetics in the range of
ms. Thus, MicroScale Thermophoresis is not
limited anymore to determine equilibrium binding
constants but can also be employed to assess
enzyme kinetics.
Jerabek-Willemsen, M., et al., Molecular Interaction Studies
Using Microscale Thermophoresis. ASSAY and Drug
Development Technologies, 2011, 9(4): 342-353.
Material and Methods
Assay conditions
For the experiment, a 110 bp DNA substrate was
pre-stained with 4 x SybrGold for 10 min at room
temperature. Labeled DNA was used at a final
concentration of 2 ng/µl. DNase I (bovine,
purchased from Roche Diagnostics GmbH) was
added at a final concentration of 0.05 mg/ml.
Samples were prepared in a buffer containing
30 mM HEPES pH 7.8, 25 mM NaCl, 1 mM DTT,
0.5 mM MgCl2 and 0.05 % Tween-20. For real
time measurements, a mastermix was prepared
on ice containing all components but the enzyme.
9 µl of the mastermix were added to two separate
tubes: one containing the enzyme (1 µl of a 10 x
stock with 0.5 mg/ml) and 2 µl H2O (for volume
adjustment), the second one containing the
enzyme and 2 µl 0.5 M EDTA (0.1 M EDTA final
concentration, serving as negative control). The
capillaries were filled very quickly and subjected to
MST measurements at room temperature.
For the quenching experiment, a mastermix was
prepared on ice. The enzyme was added last to
start the nuclease reaction and the reaction mix
was transferred to room temperature. 10 µl
aliquots of the mastermix were added to tubes
containing 2 µl 0.5 M EDTA (0.1 M EDTA final) to
stop the reaction after different incubation periods.
For both types of measurement, the samples were
filled into standard capillaries (Cat#K002).
Seidel, S. A. I., et al., Microscale Thermophoresis quantifies
biomolecular interactions under previously challenging
conditions. Methods, 2013, in press
Kunitz, M., Crystalline Desoxyribonuclease. I. Isolation and
general properties. Spectrometric method for the measurement
of desoxyribonuclease activity. J. Gen. Physiol., 1950, 33, 349362.
Vanecko, S. and Iaskowski, M., Studies of the Specificity of
Deoxyribonuclease I. III. Hydroloysis of chains carrying a
monoesterified carbon 5’. J. Biol. Chem., 1961, 236, 33123316.
© 2013 NanoTemper Technologies GmbH
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