Biomedical Engineering Reference
In-Depth Information
side. This fact indicates that uncomplexed SPG was mixed with the dA
60
/SPG
complex in the main peak. The UV showed a small secondary peak at 20 min
with the same position as dA60 alone, indicating the presence of uncomplexed
dA
60
. When this bimodal peak was deconvoluted with two Gaussians
representing dA
60
/SPG and dA
60
(dotted lines in the UV chart of
Figure 8.2A), the complexation yield was estimated to be 96%. When the
sample was prepared at a dA
60
excess composition of 3:1, the UV and RI
became less different, while the amount of uncomplexed dA was increased
along with the composition (data not shown). This result can be interpreted as
the main peak contained less free SPG and the composition was almost
uniform in the main peak. Therefore, we used the dA excess composition for
further analysis for the all phosphodiester complexes. When we carried out the
same experiment for S-dA
60
/SPG, we observed that the stoichiometric sample
had no free S-dA
60
and the complexation yield was almost 100% (Figure 8.2B).
The peak top appeared at 17 min, becoming more forward than dA
60
/SPG
(18 min) and close to tSPG (16.2 min), indicating that S-dA
60
/SPG shows a
higher complexation yield than dA
60
/SPG.
d
n
4
y
3
n
g
|
0
8.2.3
Thermal Stability of the Complexes
Since the above result can be interpreted by the S-dA
60
having a higher affinity
than dA
60
, we examined the composition dependence of the phosphor-
othioated backbone. We prepared six dA
40
samples with different phosphor-
othioate (PS) contents: 0/39 PS, 8/39 PS, 13/39 PS, 20/39 PS, 26/39 PS, and 39/
39 PS (see Table 8.1 for the substitution sites), and made the complexes with
SPG.
43
Figure 8.3A shows the CD spectra in the range 240-320 nm for all PS
and phosphodiester (PO) dA
40
samples at 20 uC. The overall spectral shapes
were almost same for all samples, although with increasing the PS content the
negative peak at 248 nm slightly increased in intensity. Since this wavelength
range is related to the induced CD between the base moieties, the similarity in
CD indicates that all dA
40
samples take the same conformation. Upon
complexation, the CD spectra are drastically changed, as presented in
Figure 8.3B, where for convenience the naked and complexed PO-dA
40
are
compared. This means that dA adopts a new conformation to form the SPG
Table 8.1
Sample codes and the sequences.
Sample code DNA sequence
a
0/39 A
40
8/39 (AsAAAA)
8
13/39 (AsAA)
13
A
20/39 (AsA)
20
26/39 (AsAsA)
13
A
39/39 (As)
39
A
a
The lower-case letter ''s'' indicates the phosphorothioate (PS) linkage.
