Incorporation of 4′-C-aminomethyl-2′-O-methylthymidine into DNA by thermophilic DNA polymerases

Article abstract of DOI:10.1039/c2cc35222b

The dual modified nucleotide 4′-C-aminomethyl-2′-O- methylthymidine 5′-triphosphate was synthesized and enzymatically incorporated into DNA by the thermophilic DNA polymerases Pfu and Therminator III. The dual ribose modification imparted increased exonuclease resistance to DNA compared to the well-known 2′-O-methyl modification.

Full text of DOI:10.1039/c2cc35222b

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COMMUNICATION
Incorporation of 40-C-aminomethyl-20-O-methylthymidine into DNA by
thermophilic DNA polymerasesw
a
a
b
a
¨
Ganesh N. Nawale, Kiran R. Gore, Claudia Hobartner* and P. I. Pradeepkumar*
Received 20th July 2012, Accepted 6th August 2012
DOI: 10.1039/c2cc35222b
The dual modified nucleotide 40-C-aminomethyl-20-O-methyl-
thymidine 50-triphosphate was synthesized and enzymatically
incorporated into DNA by the thermophilic DNA polymerases
Pfu and Therminator III. The dual ribose modification imparted
increased exonuclease resistance to DNA compared to the well-
known 20-O-methyl modification.
Fig. 1 Structures of thymidine analogs 40-C-aminomethyl-20-O-methyl
thymidine (ammT) and 20-O-methylthymidine (mT), and structure of the
target nucleotide triphosphate 1 (ammTTP).
Functional nucleic acids such as deoxyribozymes (DNA
enzymes) and aptamers are attracting interest as potential
therapeutic tools.1 In order to impart drug-like properties to
these molecules, judicious incorporation of chemical modifica-
tions is warranted.2 Site-specifically installed nucleoside modi-
fications are often deleterious at key residues in the catalytic
core of DNA enzymes or in binding motifs of aptamers.
Improved performance can be achieved by incorporating
nucleotide analogs during in vitro selection.3 This strategy
requires DNA polymerases that can efficiently incorporate
modified nucleotides, as well as copy and extend modified
DNAs with high fidelity. Though many commercially available
polymerases are known to tolerate nucleobase modifications,4
their ability to accommodate ribose modifications met with
limited success.5 In many cases, efficient utilization has been
achieved by directed evolution of polymerases.6 A particularly
challenging modification is presented by 20-O-methyl nucleo-
tides, which could only be used with engineered polymerases7
for in vitro selection of 20-O-methyl-modified aptamers.8
We have recently demonstrated advantageous properties of
the corresponding 40-C-aminomethyl-20-O-methyl-modified
uridine and cytidine nucleosides to enhance the therapeutic
potential of small interfering RNAs (siRNA).10 Inspired by
these results, we seek to select DNA enzymes that contain
amm-modified nucleotides in the catalytic core. The analo-
gously modified thymidine has been previously investigated as
modification of antisense oligonucleotides,11 but the corre-
sponding triphosphate and its enzymatic recognition have not
been reported. Gaining access to the triphosphate 1 and
exploring its competence as a substrate for DNA polymerases
are the first steps towards evolving amm-modified DNA
enzymes.
The synthesis of triphosphate 1 (Scheme 1) started from the
known 40-C-azidomethyl-modified thymidine nucleoside 2,
which was prepared from D-glucose using reported procedures.11
Nucleoside 2 was converted to the dual modified precursor 711
using a five-step procedure. To prevent alkylation of the
nucleobase, the 4-methoxybenzyl (PMB) protecting group
was installed in compound 3. Cleavage of the 20-acetyl group
generated the crude hydroxyl precursor, which was sub-
sequently methylated to obtain compound 4. After cleavage
of the PMB group by ceric ammonium nitrate (CAN), the
azide functionality of compound 5 was reduced to an amino
group by triphenylphosphine (PPh3). The amine was protected
by a trifluoroacetyl group to produce compound 6, which was
subsequently debenzylated with 10% Pd/C in EtOH. For
synthesis of the 50-triphosphate,12 the thymidine nucleoside 7
was first treated with phosphoryl chloride (POCl3) in trimethyl
phosphate, and the dichlorophosphate intermediate was
reacted in situ with tetrabutylammonium pyrophosphate.
The N-trifluoroacetyl-protected nucleotide triphosphate 8
was isolated by ion exchange chromatography and purified
by RP-HPLC. Cleavage of the trifluoroacetyl group by aqueous
Nucleotide analogs that harbour dual ribose modifications
offer special benefits in terms of nuclease resistance and target
affinity. Here we investigate dual modified 40-C-aminomethyl-
20-O-methylthymidine (ammT) in comparison to 20-O-methyl-
thymidine (mT) (Fig. 1). We report the synthesis of ammT
triphosphate 1 and its enzymatic incorporation into DNA for
potential in vitro selection applications. The primary amino group
at the 40-substituent is expected to improve nuclease resistance
and enhance the catalytic ability of DNA enzymes, as has been
reported for a number of nucleoside analogs that carry amino
acid-like functional groups in RNA-cleaving deoxyribozymes.9
a Department of Chemistry, Indian Institute of Technology Bombay,
Powai, Mumbai 400076, India. E-mail: pradeep@chem.iitb.ac.in
b Research Group Nucleic Acid Chemistry, Max Planck Institute for
Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.
¨
E-mail: claudia.hoebartner@mpibpc.mpg.de; Fax: +49 551 2011680
w Electronic supplementary information (ESI) available: Experimental
details, figures, and NMR spectra. See DOI: 10.1039/c2cc35222b
c
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Chem. Commun., 2012, 48, 9619–9621 9619

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The negative control reaction (lane 3) in the absence of dTTP
yielded the expected 30 nt product due to termination at the
templating adenosine. With this primer–template complex,
several of the tested DNA polymerases in the absence of dTTP
extended the primer significantly beyond the pause site.15 This
indicates mismatch incorporation at the templating adenosine
and calls for caution in interpreting primer extension experi-
ments with modified nucleotide triphosphates. For this reason,
we continued our experiments with Pfu, which performed
accurately in the initial screening.16 Gratifyingly, Pfu was able
to accept 1 as a substrate and incorporated ammT into full-
length modified DNA (Fig. 2b). The incorporation and the
extension of the modified oligonucleotide are rather slow
reactions (lanes 4–10); practically useful amounts of full-
length product were produced with 200 mM 1 after incubation
for up to 12 h at 72 1C. We observed batch-to-batch variability
in performance of Pfu polymerases and found that the reac-
tion is enhanced and well reproducible in the presence of
50 mM LiCl and 800 mM 1 (Fig. 2c).
Scheme 1 Synthesis of ammTTP 1. Reaction conditions: (i) 4-methoxy
benzyl chloride, DBU, CH3CN, rt, 7 h; (ii) (a) NaOMe, MeOH, rt, 3 h;
For further investigation of efficiency and selectivity of
ammT incorporation, single-nt incorporation experiments
were performed using primer P1 and template T2 (Fig. 3a,
N = A). Extension of the primer with ammT occurred
efficiently with Therminator III DNA polymerase. This
enzyme is a commercially available variant of 91 N DNA
polymerase described to incorporate 30-modified nucleotide
analogs. We found that Therminator III completely extended
the primer P1 with ammT within 10 min in the presence of
100 mM 1, which was much more efficient than Pfu. The
modified DNA was isolated and incorporation of ammT was
confirmed by ESI-MS (Fig. S1, ESIw).
(b) NaH, MeI, THF, rt,
3 h; (iii) ceric ammonium nitrate,
CH3CN–H2O (9 : 1) rt, 50 h; (iv) (a) PPh3, THF, H2O, 45 1C, 4 h,
(b) EtOCOCF3, Et3N, rt, 16 h; (v) 10% Pd/C, EtOH, 70 1C,
48 h; (vi) (a) proton sponge, PO(OMe)3, POCl3, ꢀ20 to ꢀ13 1C, 3 h,
(b) (nBu4N)2H2P2O7, nBu3N, DMF, 0 1C, 1.5 h; then Et3NH+
ꢁ
HCO3ꢀ; (vii) Et3N–MeOH–H2O 1 : 3 : 7, rt, 14 h.
NH3 under various reaction conditions was challenging due to
partial degradation of the triphosphate. Successful deprotection
was finally accomplished by treatment with NEt3–MeOH–H2O
(1 : 3 : 7).13 The target nucleotide 1 was purified by RP-HPLC
and characterized by NMR and MS.
Since 20-O-methyl-modified nucleotides are generally difficult
substrates for DNA and RNA polymerases, we examined the effect
of the 40 modification by comparing single-nt extension experi-
ments with dTTP, mTTP and ammTTP. Interestingly, mT was
also efficiently incorporated by Therminator III DNA polymerase,
The nucleoside triphosphate 1 was then studied as a sub-
strate for various DNA polymerases, including Klenow frag-
ment, Taq, Vent(exoꢀ), Pfu, KOD, Phusion, Therminator,
and Therminator III. The primer extension experiments used
the 23 nucleotide (nt) DNA primer P1 and the 35 nt template
T1 containing a single adenosine in the coding region
(Fig. 2).14 The results are here shown for Pfu polymerase. As
expected, the positive control reaction (lane 2) using the four
natural dNTPs resulted in a full-length extension product.
Fig. 2 Primer extension reactions using Pfu polymerase. (a) Partial
sequences of primer and template. (b) Lanes (1): primer only, (2): pos.
ctrl. (all dNTPs), (3): neg. ctrl. (no dTTP), 4–10: timecourse of primer
extension at 72 1C, for 3 min to 12 hours, using 0.2 mM 1, 0.03 u mLꢀ1
Pfu. (c) Lanes 1–3: same as in (b), (4): 0.8 mM 1, 0.1 u mLꢀ1 Pfu,
50 mM LiCl, 72 1C, 12 h.
Fig. 3 Single-nucleotide primer extension reactions using Therminator
III DNA polymerase. (a) Partial sequences of primer and templates;
N = A, G, C, T. (b) Concentration dependence for ammTTP (1) and
mTTP using matched template T2 (N = A). (c) Selectivity of incorpora-
tion using matched (N = A) and mismatched (N = G, C, T) templates.
All reactions run at 72 1C with 0.02 u mLꢀ1 Therminator III. p = primer.
c
9620 Chem. Commun., 2012, 48, 9619–9621
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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
nucleoside precursor 7, which was prepared in overall 19 steps
from D-glucose. We found that ammT is incorporated into
DNA by recombinant thermophilic DNA polymerases Pfu
and Therminator III. This is the first report that shows a
nucleoside analog with two functionally distinct ribose modi-
fications being a competent substrate for DNA polymerases.
For the incorporation of multiple dual-modified nucleotides,
further engineering of DNA polymerases will be required.
Mutation of recently identified gate keeper amino acids in
thermophilic DNA polymerases enabled efficient RNA
synthesis.19,20 It is conceivable that analogous mutations in
Therminator III could promote synthesis of multiply ammT- or
mT-modified DNA. The observed increased resistance against
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nucleotides such as LNA or UNA. (a) R. N. Veedu, B. Vester and
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16 All other enzymes that showed the expected stop in the neg. ctrl.
reaction (e.g. Taq and Phusion) were not able to incorporate
ammT.
17 Unspecific extension by dT occurred after the first nt (Fig. S2,
ESIw). Mis-incorporation and lack of proofreading may also
account for the unexpected read-through in neg. ctrl. experiments
as mentioned in ref. 15.
18 Incorporation of mT was also tested with Pfu on the complex
P1–T1 and was comparable to incorporation of ammT (Fig. S5, ESIw).
19 Related Therminator DNA polymerases have been shown to
polymerize ribonucleotides, but the acceptance of mNTPs
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c
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Chem. Commun., 2012, 48, 9619–9621 9621