Post-Synthesis Modification of DNA
FULL PAPER
were observed. Negative control with primer only (Lane 4,
Figure 1A) did not show annealing product due to the ab-
sence of template. On the other hand, primer extension
product with CBT-TTP (Lane 6, Figure 1A) in place of
dTTP gave a similar full-length DNA band to that of the
positive control with Klenow fragment and natural dNTPs
only (Lane 5, Figure 1A). Such results indicated that synthe-
sized CBT-TTP could be recognized as similar to dTTP by
the Klenow fragment, and incorporated into DNA. After
demonstrating the successful incorporation of CBT-TTP, we
further explored the feasibility of post-synthesis modifica-
tion of DNA. Since it is a long-term interest in our group to
develop boronic acid functionalized DNA for further appli-
cations, a 1,2-aminothiol conjugated boronic acid (Cys-BA)
probe was synthesized as an example for post-synthesis
modification (Figure 1, see Scheme S1 in the Supporting In-
formation for the synthetic route). The same 21-nt template
and 14-nt primer without the FAM label were used. As
shown from Figure 1B, fully extended product was observed
by using both dNTPs (Lane 1, Figure 1B) and CBT-TTP
(Lane 2, Figure 1B). However, after being treated with Cys-
BA (1 mm, final concentration (50 equiv) for 30 min, supple-
mented with tris(2-carboxyethyl)phosphine (TCEP, 1 mm
final concentration)) with the primer extension product,
only CBT-DNA21 (CBT incorporated DNA product, 21-nt)
showed the post-synthesis product as expected (Lane 4, Fig-
ure 1B). The reduced mobility of Cys-BA “click”-labeled
CBT-DNA21 indicated the reaction, which is presumably due
to the interaction between boronic acid and polyacrylamide
matrix.[14,21b] The result was further supported by the band
with different mobility after treating the Cys-BA “click”-la-
beled CBT-DNA21 with H2O2 (1 mm final concentration, 1 h;
Lane 5, Figure 1B), which is due to the well-known oxida-
tion reaction of converting the phenyl boronic acid function-
al group into a phenol group.[28] As a control, the mobility
for the dNTPs-DNA21 (extended DNA product by using
dNTPs, 21-nt) band did not change after treatment with
Cys-BA (Lane 3, Figure 1B), which indicated no reactions,
as expected.
product, was observed, as expected.[14,21b] Such results con-
firmed the intended click modifications. In addition, the
effect of a CBT and boronic acid moiety on the thermosta-
bility of a DNA duplex was investigated through thermode-
naturation. The results (see Figure S8 in the Supporting In-
formation) suggest that the incorporation of one CBT or
boronic acid moiety in a 21 bp DNA duplex only slightly de-
creased its stability (Tm decreased from 73.008C to 70.96
and 70.248C, respectively).
After successful incorporation of one CBT moiety into
DNA by using the Klenow fragment catalyzed primer exten-
sion reaction, we further explored the feasibility of incorpo-
rating multiple CBT moieties. Thus, 21-nt Template-2 and
Template-3 were designed to incorporate two and three
CBT-TTP, respectively. To our surprise, the primer extension
reaction catalyzed by the Klenow fragment was unsuccess-
ful, as indicated by the presence of multiple incomplete
bands, even at elevated temperature or with a longer reac-
tion time (results not shown). Knowing family B polymeras-
es are relatively more tolerant to modified TTP,[29] a family
B polymerase (KOD XL) from Thermococcus kodakaraen-
sis, which is a mixture of the natural form and an exoÀ
mutant, was thus chosen for the reaction. As shown in Fig-
ure 2A, the electrophoretic mobility of modified DNA21
Figure 2. A) Primer extension with CBT-TTP catalyzed by KOD XL
DNA polymerase, 20% PAGE analysis: 1) Template-1 with dTTP,
2) Template-1 with CBT-TTP, 3) Template-2 with CBT-TTP, 4) Template-
3 with CBT-TTP; B) Post-synthesis labeling by using extension product
of Template-3 and “click” reagent Cys-BA, 20% PAGE analysis:
1) dNTPs-DNA21, 2) CBT-DNA21, 3) CBT-DNA21 +Cys-BA.
Understandably, mobility studies alone would not be
enough to prove the post-synthesis modifications. MALDI-
MS was used to further examine the DNA products. Specifi-
cally, dNTP-DNA21 treated with Cys-BA (Lane 3, Fig-
ure 1B) had the same peak with a m/z of 6518 (Figure 1D,
calcd: 6519 [M+H]+) as the original DNA product. Such re-
sults indicate that Cys-BA does not interfere/react with
dNTP-DNA21, as expected. In contrast, full extension of the
primer by using CBT-TTP instead of dTTP yielded a CBT-
DNA21 (Lane 2, Figure 1B) with a m/z of 6716 (Figure 1C,
calcd: 6717 [M+H]+) in MALDI-MS. When treated with
Cys-BA, CBT-DNA21 was converted to a “click”-labeled
product (Lane 4, Figure 1B) with a m/z of 6917 (Figure 1E,
calcd: 6917 [MÀ2H2O+H]+), corresponding to the Cys-BA
“click”-labeled CBT-DNA21 product. After treating the
“click” product (Lane 4, Figure 1B) with H2O2, a product
(Lane 5, Figure 1B) with a m/z of 6929 (Figure 1F, calcd:
6927 [M+H]+), corresponding to the boronic acid oxidation
(Lanes 2, 3, and 4) was further decreased with the incorpo-
ration of the CBT moiety at one or more positions. This
clearly indicated the successful incorporation of multiple
CBT-TTP units into DNA through enzyme-catalyzed reac-
tions. This was further confirmed by MALDI analysis of the
incorporated product by using Templates-2 and -3 (calcd for
Template-2: 6929 [M+H]+; found: 6930; calcd for Template-
3: 7135 [M+H]+; found: 7136, see Figures S6 and S7 in the
Supporting Information). The extension product formed by
using Template-3 was further used for post-synthesis modifi-
cation by Cys-BA. As is shown in Figure 2B, the incorporat-
ed product (Lane 2) could be successfully labeled by Cys-
BA, showing a slower-moving band (Lane 3), which is con-
sistent with the phenomena observed when using the tem-
plate with incorporation of one CBT moiety.
Chem. Eur. J. 2013, 19, 4036 – 4042
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