Templated chemistry for monitoring damage and repair directly in duplex DNA

Article abstract of DOI:10.1039/c2cc34060g

We report the fluorogenic detection of the product of base excision repair (an abasic site) in a specific sequence of duplex DNA. This is achieved by DNA-templated chemistry, employing triple helix-forming probes that contain unnatural nucleobases designed to selectively recognize the site of a missing base. Light-up signals of up to 36-fold were documented, and probes could be used to monitor enzymatic removal of a damaged base.

Full text of DOI:10.1039/c2cc34060g

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COMMUNICATION
Templated chemistry for monitoring damage and repair directly in
duplex DNAwzy
Seoung Ho Lee, Shenliang Wang and Eric T. Kool*
Received 6th June 2012, Accepted 27th June 2012
DOI: 10.1039/c2cc34060g
We report the fluorogenic detection of the product of base
excision repair (an abasic site) in a specific sequence of duplex
DNA. This is achieved by DNA-templated chemistry, employing
triple helix-forming probes that contain unnatural nucleobases
designed to selectively recognize the site of a missing base.
Light-up signals of up to 36-fold were documented, and probes
could be used to monitor enzymatic removal of a damaged base.
for detection of single-stranded DNAs and RNAs, and only
one report exists of its application in fluorogenic reporting on
duplex DNA.8
Our design begins with the use of triple helices as targeting
motifs, and employs specifically designed modified bases to
recognize abasic sites within this triplex. Extensive research
has shown that pyrimidine-rich oligodeoxynucleotides can be
used to bind to purine-rich sites in duplex DNA.9 Models of
the base triads in such triple helices (Fig. 1B) suggest that
larger-than-natural bases with elongated structure might fit into
the site where a purine is missing; yet such large nucleobases
would, in principle, be sterically blocked from fitting where the
prior damaged base (or an undamaged one) exists. Finally, for
fluorogenic signaling we adopted the recently described Q-STAR
templated chemistry, in which a fluorophore-containing probe is
rendered dark by a quenching group attached via an a-azidoether
linker.7m,8 When a second probe containing a triarylphosphine
group binds adjacent, it triggers accelerated Staudinger reduction
of the azide, releasing the quencher and yielding robust fluores-
cence enhancement.
Base excision repair (BER) is a major cellular pathway for removal
of nucleobases that are damaged by hydrolysis, oxidation, or
alkylation.1,2 The immediate product of this excision is an abasic
site. Since abasic sites arise from spontaneous depurination as
well as BER, they are perhaps the most commonly found lesion
in cellular DNA, and additional cellular mechanisms exist to
replace them with coding bases.1
Base excision repair pathways are not only important in
avoidance of cellular mutations, but also are considered potential
therapeutic targets for cancer.3 Thus methods for monitoring base
excision as it occurs in DNA duplexes are of significant interest,
both for basic biological study and for evaluating potential
inhibitors of these processes. The majority of methods for
evaluating base excision involve multiple biochemical steps, and
are not amenable to high-throughput screens, or to possible
intracellular reporting.4 A few reports exist of fluorogenic assays
based on synthetic duplex DNAs containing quenchers and
fluorophores; however, efficiency has been modest, with light-up
signals of 4–8-fold,5,6 and can require multiple enzyme activities.5
Here we report the use of designed novel nucleobases coupled
with DNA-templated fluorogenic chemistry to selectively recognize
abasic sites (resulting from base excision repair) in a targeted
sequence of duplex DNA. Nucleic acid-templated chemistry has
previously been used in multiple laboratories for detection of
undamaged natural sequences of DNA or RNA.7 It has not been
used previously for detection of damaged structures and sequences.
Moreover, templated chemistry has been used almost exclusively
Department of Chemistry, Stanford University, Stanford,
California 94305, USA. E-mail: kool@stanford.edu;
Fax: +1 650 725 0259; Tel: +1 650 724 4741
w The manuscript is dedicated to the memory of H. Gobind Khorana.
z This article is part of the ‘Nucleic acids: new life, new materials’ web
themed issue.
y Electronic supplementary information (ESI) available: Detailed
experimental methods and characterization data. See DOI: 10.1039/
c2cc34060g
Fig. 1 Structures and design for recognition of abasic sites arising
from DNA repair. (A) Nucleosides I and Y, designed with imidazophen-
anthrene and pyrene nucleobase replacements respectively. (B) Diagram
showing possible fit of extended nucleobases (blue and red) from the
Hoogsteen third strand of a triplex into an abasic site in a target duplex
(adenine is shown in gray as the potential missing base).
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 8069–8071 8069

As candidate modified nucleobases for recognizing abasic sites,
we designed pyrene (Y) and imidazophenanthrene (I) (Fig. 1),
expecting that (a) their elongated geometry should allow them to
reach from the third strand into an abasic site in the purine-rich
strand of the duplex, and (b) their large size and hydrophobic
surfaces would lead to strong stacking with bases neighboring the
abasic site. The tetracyclic nucleoside I was previously unknown,
while the pyrene nucleoside Y has been studied before in other
contexts.10 The b-anomeric pyrene nucleobase has been shown to
pair selectively opposite abasic sites in single-stranded DNAs.10b
This compound has not been studied previously in triple helices,
to our knowledge. The synthesis of the imidazophenanthrene
nucleoside (I) is described in the ESI.y The second deoxyriboside
(b-pyrene, Y) was synthesized via Pd-catalyzed coupling.11 The
compounds were converted to 50-dimethoxytrityl, 30-phos-
phoramidite derivatives following standard methods.
short quenched probes containing them with the different target
duplexes. Possible binding transitions were evaluated by thermal
melting experiments; the data are shown in Fig. S6 and S7 in the
ESI.y The results show that the unmodified hairpin target has a
melting temperature (Tm) of ca. 73 1C under these conditions,
while the abasic duplexes show two high-temperature melting
transitions (B57, 68 1C), presumably due to the disruption of
contiguous stable structure by the missing base. When the 14mer
probes containing I or Y were added, a new low-temperature
transition (Tm B 10–20 1C) characteristic of third-strand binding
was seen with the abasic duplexes, but not with the intact duplex
(Fig. S6, ESIy). This provides evidence of selective binding of
the new probes with the damaged DNAs, indicating possible
abasic site recognition by the extended nucleobases. Thermal
denaturation experiments with the TPP probe also showed an
apparent triplex-binding transition at ca. 35 1C (Fig. S7, ESIy),
confirming that it binds the duplex as expected.
Oligodeoxynucleotides containing these two modified nucleo-
tides, along with naturally-substituted controls (Fig. 2), were
prepared using standard automated DNA synthesis chemistry.
The 14-nt oligomers also contained a fluorescein label on a thymine
near the 50 end, and included the quencher-linker at the 50 end.8
Oligonucleotides were purified by HPLC and were characterized
by MALDI-TOF mass spectrometry (see ESIy). For initiating
templated reactions, we also prepared a 30-conjugated triaryl-
phosphine (TPP)-containing 14mer probe,7f,m designed to bind
adjacent to the quenched probes and trigger reaction by
juxtaposing the phosphine with the quencher/azidolinker
group. A final modification employed in all third-strand
probes was the use of pseudoisocytosine (c) in place of
cytosine; this substitution enables triplexes to form stably at
neutral pH.12 A number of 28 bp duplex DNAs were prepared
as targets, and contained either a uracil (a model damaged
base) at the position being probed, an undamaged adenine, a
tetrahydrofuran abasic analog (THF),13 or a native abasic
sugar (Fig. 2).14 The targets were constructed as hairpins to
render them stable and to fix the stoichiometry of the strands.
First we evaluated the binding properties of the modified
nucleobases, by looking for evidence of hybridization of the
We then proceeded to test whether the probes could carry out
selective templated chemistry to yield fluorescence signals with
abasic target DNAs. Initial experiments were performed with
200 nM target DNA and 200 nM Q-STAR probe, with an excess
of phosphine probe (600 nM) to compensate for any phosphine
oxidation that might occur during the reaction.7e Time courses of
fluorescence emission signals for the reactions at 25 1C are shown
in Fig. 3. The data show that both modified probes yield clear
fluorogenic signals that increase over 3–4 hours in the presence of
target DNA containing either the THF abasic site or the native
abasic site. Signals increase by robust factors of 15–18-fold after
3.5 h (probe I) and 18–20-fold (probe Y); extended experiments
demonstrate up to 36-fold enhancement after 12 h (Fig. S8, ESIy).
The reactions are clearly templated by the target duplexes, since
controls omitting duplexes yielded only a very small background
signal after 3.5 h (Fig. 3). Importantly, the reaction is strongly
abasic-selective, as the intact adenine-containing target yielded
little enhancement (ca. 1.5 fold) after this time. Extended reaction
times with excess probes also revealed a small degree of isothermal
amplification due to slow turnover of the probes on the target
(Fig. S9, ESIy). Overall, the results show that the probes 1 and 2,
containing the unnatural bases I and Y, respectively, can efficiently
and selectively recognize missing bases in DNA and report on it
with a fluorogenic signal.
Finally, we explicitly tested whether enzymatic repair could be
probed by this approach, using uracil, the deaminated product
arising from cytosine hydrolysis, as the damaged base. We
targeted a duplex containing a U–G mismatch at the variable
position, with U situated in the purine-rich strand. Uracil DNA
glycosylase (UDG) enzymes remove uracil from such mismatched
pairs, leaving abasic sites in their place.15 Therefore we tested
probes both in the presence of the original U–G mismatch and in
the presence of added E. coli UDG enzyme (see Fig. S10, ESIy).
Control experiments using the Y-containing Q-STAR probe with
the U–G mismatch DNA or with an A–T pair showed only
relatively small signals over a 3.5 h time course, confirming that a
correct pair or even a mismatched (damaged) base sterically blocks
the binding of the unnatural probe. However, when UDG was
incubated with the DNA for 30 min and then probes were added,
a robust fluorescent signal developed (14-fold enhancement with
the Y probe in 3.5 h and 10-fold for the I probe), consistent with
the prior results with chemically synthesized abasic DNAs.
Fig. 2 Probe sequences and structures. Targets are 28 bp hairpin
duplexes. Phosphine probe carries triarylphosphine conjugated to the
30-end as shown. Quenched (Q-STAR) probe is labeled with fluorescein
and is conjugated at the 50-end with a quencher-linker containing a
reductively cleavable azidoether group. cC is pseudoisocytidine, used
for triplex formation at neutral pH.
c
8070 Chem. Commun., 2012, 48, 8069–8071
This journal is The Royal Society of Chemistry 2012

Notes and references
1 J. Lhomme, J. F. Constant and M. Demeunynck, Biopolymers,
1999, 52, 65.
2 (a) L. B. Meira, N. E. Burgis and L. D. Samson, Adv. Exp. Med.
Biol., 2005, 570, 125; (b) K. Hitomi, S. Iwai and J. A. Tainer,
DNA Repair, 2007, 6, 410; (c) J. C. Fromme, A. Banerjee and
G. L. Verdine, Curr. Opin. Struct. Biol., 2004, 14, 43.
3 C. K. Anders, E. P. Winer, J. M. Ford, R. Dent, D. P. Silver,
G. W. Sledge and L. A. Carey, Clin. Cancer Res., 2010, 16, 4702.
4 G. L. Dianov, Toxicology, 2003, 193, 35–41.
5 A. C. Drohat, J. Jagadeesh, E. Ferguson and J. T. Stivers,
Biochemistry, 1999, 38, 11866.
6 (a) A. Maksimenko, A. A. Ishchenko, G. Sanz, J. Laval,
R. H. Elder and M. K. Saparbaev, Biochem. Biophys. Res. Commun.,
2004, 19, 240; (b) B. Liu, X. Yang, K. Wang, W. Tan, H. Li and
H. Tang, Anal. Biochem., 2007, 366, 237; (c) Y. Kim and I. S. Hong,
Bull. Korean Chem. Soc., 2009, 30, 2149.
7 (a) A. Oser and G. Valet, Angew. Chem., Int. Ed., 1990, 102, 1197;
(b) Y. Xu, N. B. Karalkar and E. T. Kool, Nat. Biotechnol., 2001,
19, 148; (c) A. Mattes and O. Seitz, Chem. Commun., 2001, 21, 2050;
(d) S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 2096;
(e) J. Cai, X. Li, X. Yue and J.-S. Taylor, J. Am. Chem. Soc., 2004,
126, 16324; (f) K. Sakurai, T. M. Snyder and D. R. Liu, J. Am. Chem.
Soc., 2005, 127, 1660; (g) T. N. Grossmann and O. Seitz, J. Am.
Chem. Soc., 2006, 128, 15596; (h) Z. L. Pianowski and N. Winssinger,
Chem. Commun., 2007, 3820; (i) H. Abe, J. Wang, K. Furukawa,
K. Oki, M. Uda, S. Tsuneda and Y. Ito, Bioconjugate Chem., 2008,
19, 1219; (j) R. M. Franzini and E. T. Kool, ChemBioChem, 2008,
9, 2981; (k) Y. Huang and J. M. Coull, J. Am. Chem. Soc., 2008,
130, 3238; (l) T. N. Grossmann, A. Strohbach and O. Seitz,
ChemBioChem, 2008, 9, 2185; (m) R. M. Franzini and E. T. Kool,
J. Am. Chem. Soc., 2009, 131, 16021; (n) Z. L. Pianowski, K. Gorska,
L. Oswald, C. A. Merten and N. Winssinger, J. Am. Chem. Soc.,
2009, 131, 6492; (o) K. Furukawa, H. Abe, K. Hibino, Y. Sako,
S. Tsuneda and Y. Ito, Bioconjugate Chem., 2009, 20, 1026.
8 H. Li, R. M. Franzini, C. Bruner and E. T. Kool, ChemBioChem,
2010, 11, 2132.
Fig. 3 Fluorogenic signaling of missing bases by probes containing I
(A) and Y (B). Positive signals are seen for either a natural abasic site or
the tetrahydrofuran (THF) abasic site mimic. Conditions: 200 nM target
9 (a) H. E. Moser and P. B. Dervan, Science, 1987, 238, 645;
(b) N. T. Thuong and C. Helene, Angew. Chem., Int. Ed., 1993,
´
105, 697; (c) M. D. Frank-Kamenetskii and S. M. Mirkin, Annu.
Rev. Biochem., 1995, 64, 65.
DNA and fluorogenic probes; 600 nM TPP trigger probe; 70 mM Na+
,
10 mM Mg2+, 25 1C. Excitation 490 nm; emission 525 nm.
10 (a) R. X Ren, N. C. Chaudhuri, P. L. Paris, S. Rumney and
E. T. Kool, J. Am. Chem. Soc., 1996, 118, 7671; (b) T. J. Matray
and E. T. Kool, J. Am. Chem. Soc., 1998, 120, 6191; (c) T. J. Matray
and E. T. Kool, Nature, 1999, 399, 704; (d) Y. L. Jiang, K. Kwon
and J. T. Stivers, J. Biol. Chem., 2001, 276, 42347; (e) Y. Cho and
E. T. Kool, ChemBioChem, 2006, 7, 669.
11 (a) A. F. Littke and G. C. Fu, J. Org. Chem., 1999, 64, 10;
(b) J. Guo, S. Wang, N. Dai, Y. N. Teo and E. T. Kool, Proc.
Natl. Acad. Sci. U. S. A., 2011, 108, 3493.
´
12 A. Ono, P. O. P. Tso and L. S. Kan, J. Am. Chem. Soc., 1991, 113, 4032.
13 M. Takeshita, C. N. Chang, F. Johnson, S. Will and
A. P. Grollman, J. Biol. Chem., 1987, 262, 10171.
14 K. Groebke and C. J. Leumann, Helv. Chim. Acta, 1990, 73, 608.
15 L. H. Pearl, Mutat. Res., 2000, 460, 165.
16 J. Gong and S. J. Sturla, J. Am. Chem. Soc., 2007, 129, 4882.
17 (a) S. Rosa, P. Fortini, P. Karran, M. Bignami and E. Dogliotti,
Nucleic Acids Res., 1991, 19, 5569; (b) H. Ide, K. Akamatsu,
Y. Kimura, K. Michiue, K. Makino, A. Asaeda, Y. Takamori and
K. Kubo, Biochemistry, 1993, 32, 8276; (c) M. Adamczyk,
P. G. Mattingly and J. A. Moore, Bioorg. Med. Chem. Lett., 1998,
8, 3599; (d) M. Adamczyk, P. G. Mattingly, J. A. Moore and Y. Pan,
Org. Lett., 1999, 1, 779; (e) S. Dhar, T. Kodama and
M. M. Greenberg, J. Am. Chem. Soc., 2007, 129, 8702; (f) Y. Wang,
L. Liu, C. Wu, A. Bulgar, E. Somoza, W. Zhu and S. L. Gerson, Nucl.
Med. Biol., 2009, 36, 975; (g) N. Kojima, T. Takebayashi, A. Mikami,
E. Ohtsuka and Y. Komatsu, J. Am. Chem. Soc., 2009, 131, 13208;
(h) M. A. Fakhari and S. E. Rokita, Chem. Commun., 2011, 47, 4222.
18 G. Chowdhury and F. P. Guengerich, Chem. Res. Toxicol., 2009,
22, 1310.
Thus the preliminary experiments show that probes 1 and 2 can
detect the enzymatic repair of uracil-containing DNAs that leads
to abasic sites formed in situ. It is worth noting that the abasic
site is an intermediate in base excision repair of multiple forms of
DNA damage, and thus we speculate that the current probes
may be useful as reporters for diverse enzymatic pathways.
These results provide proof-of-principle that designed nucleo-
bases can be used to recognize the abasic product of base excision
repair located directly in duplex DNA. Previous researchers have
described modified nucleobases that can recognize a damaged
nucleobase,16 but in the context of single-stranded DNA only.
Since damage and its repair occur naturally in the double-stranded
context, the current approach offers a biologically relevant strategy
for detecting them. In addition, we show that this highly selective
recognition can be coupled to a fluorogenic reporting process by
applying templated chemistry to this recognition. Although abasic
sites have been detected by many approaches previously,17–19 few
have yielded a fluorogenic signal, which simplifies detection by
requiring only one step.
We envision this approach as being potentially useful for basic
science studies of BER, using pre-made triplex target DNAs as
engineered sites of repair. Such probe damaged duplexes, combined
with our fluorogenic probes, could also be used in high-throughput
screens for inhibitors of various BER enzymes.
19 (a) M. C. Buzzeo and J. K. Barton, Bioconjugate Chem., 2008,
19, 2110; (b) U. Jakobsen, S. A. Shelke, S. Vogel and S. T.
Sigurdsson, J. Am. Chem. Soc., 2010, 132, 10424; (c) A. A. Tanpure
and S. G. Srivatsan, Chem.–Eur. J., 2011, 17, 12820.
This work was supported by the U.S. National Institutes of
Health (GM068122).
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 8069–8071 8071