©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
3
APPLICATION NOTE
0 5 10 15 20
0.70
0.75
0.80
0.85
0.90
0.95
1.00
Relative
Fluorescence
[-]
10
-9
HSA [M]
890
900
910
920
930
940
Fnorm
[‰]
10
-8
10
-7
10
-6
10
-5
10
-4
10
-3
10
-2
0 5 10 15 20
MST experiment time [s]
0.7
0.8
0.9
1.0
Relative
Fluorescence
[-]
885
890
895
Fnorm
[
‰]
MST experiment time [s]
10
-9
HSA [M]
10
-8
10
-7
10
-6
10
-5
10
-4
10
-3
10
-2
and in solution with only minute amounts of
fluorescently-labeled oligonucleotides or proteins. And
unlike other approaches, a K
d
can be measured in less
than 10 min using Monolith NT.115, or less than 2 min
using the Monolith NT.Automated.
Results and discussion
In order to avoid modification of HSA by labeling with
a fluorophore that could allosterically influence the
binding of the oligonucleotides, we decided to label the
oligonucleotide with Cy5 instead. This approach also
means one can use the labelled oligonucleotide in a
competition assay with any unlabeled oligonucleotide.
Phosphate buffer saline (PBS) was used as the buffer
for the MST assays. Because Tween-20 interacts with
HSA
6
, we performed the binding experiments with
and without 0.05% Tween-20 in the assay buffer. The
addition of Tween-20 to PBS resulted in over three-
fold worsening of the K
d
value for the Cy5-modified
phosphorothioate oligonucleotide. Consequently,
all experiments were performed in the absence of
Tween-20. The K
d
value determined was 64.9 ± 2.7 µM
(Figure 1, le panel). Interestingly, no binding of the
Cy5-cholesterol-conjugated oligonucleotide could
be determined in the absence of Tween-20. Upon
the addition of 0.05% Tween-20, the binding of this
oligomer to HSA could be determined with the K
d
value
of 28.4 ± 3.8 µM (Figure 1, right panel). We hypothesize
that Tween-20 binds to the fatty acid binding pocket
8
causing the conformational change, which in turn
allosterically enhances the binding of the Cy5-
cholesterol-conjugated oligonucleotide to HSA.
Figure 1: The Cy-5 labeled nucleotides bind to HSA. For the experiments 20 nM of labeled nucleotide was used and HSA titrated at indicated concentrations. Le panel:
Cy5- modified phosphorothioate oligonucleotide, K
d
= 64.9 ± 2.7 µM; right panel: Cy5-cholesterol-conjugated oligonucleotide, K
d
= 28.4 ± 3.8 µM.
