nuclease degradation makes ammT-modified DNA a promising
candidate for further investigation towards in vitro selection of
functional nucleic acids.
Financial support from the Department of Science and
Technology (DST), Government of India (FAST track
scheme), the German Academic Exchange Program (DAAD),
and the Max Planck Society is gratefully acknowledged. PIP is
a recipient of a Max-Planck India Fellowship. GN thanks
CSIR, and KG thanks UGC for research fellowships.
Notes and references
Fig. 4 (a) Extension of modified primers with 200 mM dNTPs and
0.02 u mLꢀ1 Therminator III DNA polymerase for 1 h (*6 h) at 72 1C.
(b) SVPD digestion of unmodified DNA in comparison to mT- and
ammT-terminated DNA. SVPD 10ꢀ4 u mLꢀ1, 1 mM Mg2+, 22 1C, time
points up to 5 h (experiments up to 18 h are shown in Fig. S4, ESIw).
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even at lower concentrations than ammT (Fig. 3b). The primer
was fully extended with only 1 mM mTTP in 5 min (Fig. S2,
ESIw). While the primer extension with dTTP did not stop
efficiently after the first nt extension,17 mT was only incorpo-
rated once.18
We also investigated mismatch extension of primer P1 with
thymidine and its analogs ammT and mT, using templates
T3–T5, which each contain a different nucleotide directly after
the primer (Fig. 3c). Using two different concentrations of
mTTP and ammTTP, we found that ammT was incorporated
only with the matched template T2, while mT was also
incorporated with all other templates, demonstrating low
specificity of Therminator III with 20-O-methylated analogs
(Fig. S2, ESIw). When tested with all mNTPs, Therminator III
was able to extend the primer by 6–7 methylated nucleotides
(Fig. S3, ESIw).19 DNA with 30-terminal ammT and mT could
be further extended with unmodified nucleotides to give a full-
length product (Fig. 4a). The extension reaction was slower
than the incorporation of the modification. While the
mT-modified primer was fully extended within 1 h at 72 1C, up
to 6 h were needed for full extension of ammT-terminated DNA.
The ammT- and mT-modified DNAs were characterized
with respect to their resistance against 30-exonuclease degra-
dation, using snake venom phosphodiesterase (SVPD). Inter-
estingly, the ammT-modified DNA showed improved stability
compared to mT-modified DNA, which was already more
stable than unmodified DNA (Fig. 4b). Similar results were
found for the internally ammT-modified DNA generated by
Pfu (Fig. S4, ESIw).
In summary, we have synthesized ammTTP 1 from the
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from D-glucose. We found that ammT is incorporated into
DNA by recombinant thermophilic DNA polymerases Pfu
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mT-modified DNA. The observed increased resistance against
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18 Incorporation of mT was also tested with Pfu on the complex
P1–T1 and was comparable to incorporation of ammT (Fig. S5, ESIw).
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 9619–9621 9621