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For this purpose, we designed a double-stranded DNA sequence
instead of a (G:C) pair. The second duplex contained the quencher
group at one nucleotide shift with respect to the benzyl-20-deoxy-
guanosine in the opposite sequence to maintain complementarity,
expecting that the fluorescence extinction would remain efficient
and intending to minimize the steric impediments for the repair
action of hAGT.
First, to measure the effect of these modifications in the duplex
formation, the thermal denaturation curves of all the modified and
unmodified duplexes were studied at 260 nm (see Table S2 and
Fig. S2 at Supplementary Data). In general, we observed destabi-
lization of the duplex in the presence of the bulky substituent in
the O6 position of the central guanine, compared to the controls.
However, both duplexes are stable at 30 °C. The duplex structures
of both pairs were studied by CD spectra at 25 °C confirming that
the fluorescein-benzyl substitution did not affect the overall sec-
ondary structure of the DNA (see Fig. S1, SD). Based on these
results we decided to perform the hAGT activity assays at 25 °C
that is below the melting temperature to ensure that the oligonu-
cleotides are in the duplex form.
containing a fluorophore–quencher pair (Scheme 1). The fluo-
rophore was post-synthetically and covalently attached to a mod-
ified O6-benzylguanine (FdG) and the quencher was introduced in a
very close position of the complementary strand of the duplex
(QdU-complementary or QdU-mismatch), quenching the fluores-
cence. The fluorophore was transferred together with the benzyl
group to hAGT’s active site when the protein repaired the DNA,
restoring the guanine. The removal of the fluorophore brought it
apart from the quencher, producing a significant increase in fluo-
rescence which allowed to measure the repair reaction of hAGT.
The BzdG oligonucleotide was prepared using the appropriate
modified 20-deoxyguanosine phosphoramidite (7) in the auto-
mated DNA synthesizer using standard protocols.37
Derivative 7 was prepared from 20-deoxyguanosine and the pro-
tected
N-(4-(hydroxymethyl)benzyl)-trifluoroacetamide
(1)
(Scheme 2). Its incorporation at the O6 position of the guanine
was performed through a Mitsunobu reaction. The guanine amino
function was protected with a dimethylformamidine protecting
group. Finally, the protection and functionalization of the two
hydroxyl groups were carried out under standard conditions.
The O6-benzyl-20-deoxyguanosine-containing oligonucleotide
and its complementary strands with a quencher group (Dabcyl,
Q) either at the complementary position or at one nucleotide shift
were synthesized, purified by HPLC and characterized by MALDI-
TOF (see Supplementary Data). Subsequently, the fluorescein label
(F) was attached to the O6-benzyl-20-deoxyguanosine amino group
by the fluorescein isothiocyanate (FITC) reaction.38 The DNA
duplexes, one complementary and the other containing a mis-
match due to the positioning of the dU-quencher phosphoramidite
pairing with the modified FdG, were prepared from the fluo-
rophore–quencher pair of oligonucleotides under proper annealing
conditions (Table 1). We explored to different positions of the
quencher group in the complementary sequences to ensure the
maximal quenching of fluorescence and to minimize steric effects
and complementary discrepancies. In the case of the first duplex
the placement of the quencher in the complementary base of the
modified guanine provokes a mismatch in the sequence (G:dU)
Efficient reaction with hAGT requires the FdG modification to
correctly accommodate into the active site. This brings the CH2
which is attached at the O6 position in closer proximity to the thiol
group of Cys145, making the reaction possible. To study the capac-
ity of the fluorescein-benzyl group to enter the active site of hAGT
without affecting the hAGT activity, the reaction was analyzed by
HPLC. For this purpose, the full-length hAGT was over-expressed
and purified as previously described.35,39 Increasing concentrations
of the protein were incubated with an excess of the double-
stranded FdG sequence for 90 min at 25 °C and analyzed using
HPLC. The reaction was performed in substoichiometric conditions
due to the fact that high amount of DNA was required to detect the
peak corresponding to the repaired sequence. Figure 1 shows one
of the HPLC profiles of the reaction’s final products of hAGT. The
appearance of a peak with a shorter retention time corresponds
to the repaired sequence, formed by the removal of the fluores-
cein-benzyl group. Even if the reaction was not quantitative due
to the lower amount of hAGT used in the experiment, the repair
of the alkylguanine indicated that hAGT has the capacity to
Scheme 1. (A) Chemical structure of the O6-benzylguanine with the fluorophore covalently attached to the alkyl group. (B) Chemical structure of the quencher group
(Dabcyl), covalently attached to a 20-deoxyuridine. (C) Schematic representation of the fluorescence assay. (1) Annealing of the two strands containing a fluorophore and a
quencher group, with the correspondent extinction of fluorescence. (2) hAGT repair activity over the benzylguanine, dragging the fluorescein group and significantly
increasing fluorescence.