A. Goubet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 761–763
763
incorporate seven consecutive EBNA-T nucleotides and annealed to
a 50-FAM-labeled 19 nucleotide (nt) primer P1 (Table 1).
(Fig. 5a). First, a 21 nt probe DNA complementary to the 30-end
of the primer extended top strand of the extension product was
immobilized to a streptavidin containing magnetic bead. Next,
the crude primer extension reaction mixture was subjected to phe-
nol–chloroform extraction and the aqueous phase containing DNA
was heat dissociated and immediately mixed with the magnetic
bead immobilized capture probe DNA. The resulting mixture was
gently heated at 55–60 °C and collected the clean primer extended
EBNA-T containing ssDNA while capturing the bead immobilized
capture probe on the magnet. PCR amplification was performed
using the purified primer extension product with three EBNA-T
nucleotides furnished by KOD DNA polymerase from template
T2. Primers P1 and P2 (Table 1) were used as forward and reverse
primers respectively. Phusion DNA polymerase yielded the PCR
product in good yields (Fig. 5b).
In summary, we have demonstrated that KOD DNA polymerase
is an efficient enzyme to accept EBNA-T nucleotide as a substrate.
Klenow and Phusion DNA polymerases can also incorporate EBNA-
T nucleotides. Phusion DNA polymerase efficiently amplified an
EBNA-T modified DNA template by PCR. The results reported here
clearly suggest that EBNA-T nucleotide can now be used in SELEX
processes in deriving functional nucleic acids such as aptamers
with additional functionality.
The experiment revealed that KOD (Fig. 2a), and Klenow
(Fig. S1a), DNA polymerases successfully extended the primer to
full-length whereas Phusion and Taq DNA polymerase could only
incorporate up to three and one EBNA-T nucleotide respectively
(Fig. S1b and c). KOD DNA polymerase was found to be the most
efficient one as it yielded the full-length product in good yields.
Interestingly, we observed a shift in the migration of products
between the positive control reaction using all natural nucleotides
and EBNA-T containing reaction product. This might be due to the
successive incorporation of the bulky EBNA moiety affecting the
mobility of the modified DNA. An extension experiment was also
performed to check the fidelity of EBNA-T incorporation using
other four individual natural nucleotides by KOD DNA polymerase.
The results showed that the polymerase followed the necessary
fidelity in recognizing EBNA-T nucleotides (Fig. 2b).
The stability of EBNA-T containing extension product was com-
pared with the product containing natural nucleotides by perform-
ing an extension experiment using template T1. KOD DNA
polymerase was used for this experiment as it has a very high de-
gree of 30?50 exonuclease activity. The reactions mixtures were
incubated at 72 °C and monitored at 50 s, 1, 2, 5 and 10 min. Exten-
sion product with natural nucleotide was degraded in 2 min
whereas the product containing EBNA-T was still present even at
10 min of incubation (Fig. 2c and d). Later, we investigated the suc-
cessive incorporation of EBNA-T nucleotides using a long DNA tem-
plate, T2 in presence of other three natural nucleotides. In parallel
to the positive control and the EBNA-T reactions, a negative control
reaction that lacks ‘T’ nucleotide in the dNTP mixture was also per-
formed. The designed template T2 (Table 1) directs eight consecu-
tive sites of incorporations. KOD and Phusion DNA polymerase
incorporated eight EBNA-T nucleotides successively and extended
the primer onwards to full-length (Fig. 3a and b). Notably, both
Klenow and Taq DNA polymerase failed to extend the primer after
the first EBNA-T incorporations (data not shown). As shift in the
migration of EBNA-T containing product was again observed in line
to the reaction using template T1.
Acknowledgment
This work was supported by the UQ Fellowship scheme
awarded to RNV.
Supplementary data
Supplementary data (supporting information with additional
gel images and experimental protocols) associated with this article
References and notes
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Another extension experiment was conducted with the aim of
generating EBNA-T modified DNA as a template for polymerase
chain reaction amplification (PCR) as it is the first enzymatic step
involved in the aptamer selection process. For this experiment,
the designed template T3 (Table 1) directs three EBNA-T nucleotide
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To isolate the primer extended ssDNA containing EBNA-T nucle-
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Goubet, Astrid
