COMMUNICATION
Nucleotide insertion and bypass synthesis of pyrene- and
BODIPY-modified oligonucleotides by DNA polymerasesw
Claudia Wanninger-Weiß,a Francesca Di Pasquale,b Thomas Ehrenschwender,a
Andreas Marx*b and Hans-Achim Wagenknecht*a
Received (in Cambridge, UK) 20th November 2007, Accepted 7th January 2008
First published as an Advance Article on the web 29th January 2008
DOI: 10.1039/b718002k
The chromophores pyrene and bordipyrromethenylbenzene
directly linked to the 5-position of uridine are tolerated and
recognized as thymine derivatives by DNA polymerases in
primer extension experiments.
First we investigated the Klenow fragment (exo-) of E. coli
DNA polymerase I (KF-) in its propensity to insert a nucleotide
opposite the modified DNA nucleobase. Gel electrophoretic
analysis of the radiometric primer extension reactions revealed
that the canonical bases are predominantly incorporated, that
means A opposite to 1PydU, 2PydU and BodU, and C opposite
to PydG (Fig. 1). Only minor amounts of misincorporation of G
opposite to 2PydU and less opposite to 1PydU were observed.
When all four dNTPs are present in the primer extension
experiment, KF- is able to bypass all three types of uridine
modifications (1PydU, 2PydU, and BodU) but not the modified
guanosine (PydG). This is a remarkable result since the steric
hindrance by the chromophores, especially by the bordipyrro-
methenylphenyl substituent, was expected to be significant.
Subsequently, human DNA polymerase b (Pol b), a member
of the DNA polymerase X family involved in DNA repair, and
DNA polymerase Dpo4, a representative of the Y-family, were
examined (Fig. 1). In the single nucleotide insertion experi-
ments both enzymes placed the canonical nucleotides opposite
the modification sites, but Pol b was unable to incorporate any
nucleotide opposite PydG. In contrast to KF-, a significant
amount of misincorporation was not observed. However, both
enzymes, Pol b and Dpo4, were only able to bypass the
modified uridines (except 1PydU with Pol b) in experiments
with all four dNTPs using higher polymerase concentrations
and using an extended incubation time of 60 min. Even under
these conditions, PydG could not be bypassed by any of the
polymerases (Fig. S4–S5, ESIw). The reason for this might be
that the pyrene at the 8-position induces the syn-conformation5
of the nucleotide, yielding altered base pairing properties.
Since KF- was capable of bypassing DNA template modi-
fications when all four dNTPs were present, we measured the
activity of the enzyme on the respective templates in comparison
to the unmodified template (Table 1). We employed an assay
previously established to measure DNA polymerase activity on
non-natural DNA primer template complexes.9 The data show
that the chemical modifications significantly impair bypass effi-
ciency. These effects are most pronounced when PydG was used.
The C5 modifications at pyrimidines are somewhat better toler-
ated as has been observed with other modifications before.10
Finally, we examined the absorption (Fig. S1–S3, ESIw) and
fluorescence properties (Fig. 2) of the chromophore-uridine
modified template–primer duplex in comparison with the
synthetic full-length duplex. Additionally, an oligonucleotide
was synthesized that contained the primer sequence and an
additional A as counterbase to the chromophore-modified
If fluorophores are attached to DNA bases for oligonucleotide
labeling,1 an alkyl chain linker is inserted between the chro-
mophore and DNA base to allow the replication by DNA
polymerases. However, the direct covalent attachment of
chromophores to DNA bases yields unique optical properties,
such as solvatochromism and exciplex-type emission2 that are
suitable for DNA probing. A critical issue about this direct
linkage is the question if the canonical base recognition
complementarity persists in DNA polymerase-catalyzed pri-
mer extension experiments.3 For instance, fluorophore-labeled
nucleosides and fluorosides can be applied as substrates for the
DNA polymerase.4
Over the past years, we attached synthetically pyrene5–7 or
ethynylpyrene8, for example, to DNA bases for electron
transfer studies and as fluorescent probes for DNA. To gain
more insight into the counterbase selectivity, we performed
primer extension experiments with a representative set of
modified oligonucleotides (Scheme 1). The templates con-
tained 5-(pyren-1-yl)-20-deoxyuridine (1PydU),5 5-(pyren-2-
yl)-20-deoxyuridine (2PydU),7 5-[4-(2,6-diethyl-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a-4a-diaza-s-indacyl)phenyl]-20-de-
oxyuridine (BodU) or 8-(pyren-1-yl)-20-deoxyguanosine
(PydG)6 as single modifications. The length of the radio-
actively labeled primer was chosen such that the modified
nucleotide in the template strand codes for the first nucleotide
during primer extension. Single-base incorporations were per-
formed with each of the four dNTPs exclusively to get
information about the insertion selectivity opposite to the
modified nucleotide. In addition, experiments employing all
four dNTPs simultaneously were performed to study the
elongation bypassing the modification site.
a University of Regensburg, Institute for Organic Chemistry, D-93040
Regensburg, Germany. E-mail: achim.wagenknecht@chemie.uni-
regensburg.de; Fax: +49 941-943-4617; Tel: +49 941-943-4802
b University of Konstanz, Department of Chemistry, D-78457
Konstanz, Germany. E-mail: andreas.marx@uni-konstanz.de; Fax:
+49 7531-88-5140; Tel: +49 7531-88-5139
w Electronic supplementary information (ESI) available: Synthesis,
spectra and characterization details; primer extension experiments;
DNA polymerase activity determination; UV absorption spectra. See
DOI: 10.1039/b718002k
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1443–1445 | 1443
Wanninger-Weiss, Claudia
