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4
APPLICATION NOTE
Figure 2: Comparison of the dose-response curve of both Cy5-labeled
nucleotides (A). The Cy5- modified phosphorothioate oligonucleotide yields
nearly four-fold larger amplitude and better signal-to-noise ratio than the
Cy5-cholesterol-conjugated oligonucleotide (B).
Figure 3: Competition assay between the Cy5-modified phosphorothioate
oligonucleotide and the ligands modified phosphorothioate and cholesterol-
conjugated oligonucleotides. HSA (100 µM) was prelabeled with the Cy5-
fully modified phosphorothioate oligonucleotide (50 nM) and the unlabeled
ligand titrated at indicated concentrations. The EC50 values determined were
8.6 ± 1.0 µM for the modified phosphorothioate oligonucleotide, and 3.8 ± 0.4
µM for the cholesterol-conjugated oligonucleotide.
The comparison of binding profiles of the Cy5-labeled
oligonucleotides is shown in Figure 2. Since the Cy5-
modified phosphorothioate oligonucleotide offers a
nearly four-fold increase in signal amplitude and better
signal-to-noise ratio compared to the Cy5-cholesterol-
conjugated oligonucleotide it was chosen for further
competition experiments.
For the competition assay HSA (100 µM) was
prelabeled with the Cy5-modified phosphorothioate
oligonucleotide (50 nM) for 15 min. Then, either the
unlabeled phosphorothioate modified oligonucleotide
-25
-20
-15
-10
-5
0
Δ
FNorm
[‰]
Ligand Concentration [M]
10
-9
10
-8
10
-7
10
-6
10
-5
10
-4
10
-3
modified phosphorothioate oligonucleotide
cholesterol-conjugated oligonucleotide
or cholesterol-conjugated oligonucleotide were
added in increasing concentrations and incubated
for an additional 15 min to displace the labeled
oligonucleotide from the binding pocket in PBS
without Tween-20. Both unlabeled oligonucleotides
readily displaced the Cy5-modified phosphorothioate
oligonucleotide (Figure 3). This is reflected by the EC50
values: 8.6 ± 1.0 µM for the phosphorothioate modified
oligonucleotide, and 3.8 ± 0.4 µM for the cholesterol-
conjugated oligonucleotide.
Overall, the use of Cy5-labeled oligonucleotide as
a tracer is an excellent approach to quantify the
oligonucleotide binding properties to HSA. All MST
experiments described here are characterized by an
excellent signal-to-noise ratio that enables reliable
determination of affinities between the oligonucleotides
and HSA.
A
Cy-5 labeled nucleotide Amplitude
Signal-to-
noise
Phosphorothioate
oligonucleotide
49.8 137.9
Cholesterol-conjugated
oligonucleotide
13.7 28.1
B
HSA [M]
0
10
20
30
40
50
Δ
FNorm
[‰]
10
-9
10
-8
10
-7
10
-6
10
-5
10
-4
10
-3
10
-2
Cy5-modified phosphorothioate oligonucleotide
Cy5-cholesterol-conjugated oligonucleotide
