A Synthetic Nucleoside Probe that Discerns a DNA Adduct from Unmodified DNA
Jiachang Gong and Shana J. Sturla*
Department of Medicinal Chemistry and The Cancer Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455
Received January 30, 2007; E-mail: firstname.lastname@example.org
Biologically reactive chemicals alkylate DNA and induce
structural modifications in the form of covalent adducts.1Certain
bulky DNA adducts can persist, escape repair, and serve as
templates for polymerase-mediated DNA synthesis, resulting in
mutation and cancer.2Correlating chemical structures and quantita-
tive levels of adducts with toxicity is central to understanding
chemical mechanisms of carcinogenesis for specific agents. Major
challenges include that DNA adducts are formed at exceedingly
low levels, adduct mixtures are often formed, and minor lesions
may have greater biological impact than more abundant products.2
New molecular approaches for addressing specific low-abundance
adducts are needed, and we describe here the first example of a
synthetic nucleoside that may serve as the chemical basis for a probe
of a bulky carcinogen-DNA adduct.
Dozens of thermodynamically stable synthetic base pairs have been
reported3and continue to emerge as powerful tools in areas such as
polymerase fidelity,4DNA helix stability,5nucleic acids with novel
functionality,6and expanded genetic systems,3to cite selected exam-
ples. Recently, Hirao and co-workers successfully have amplified
an entirely synthetic base pair.7Amplified in a polymerase-mediated
process or used in hybridization-based strategies, synthetic nucleo-
sides might act as probes of DNA damage, but to our knowledge,
no examples of synthetic nucleosides that pair selectively with an
adduct generated in a natural physiological system are known.
O6-Benzyldeoxyguanosine (1, O6-BnG; Figure 1) is a bulky DNA
adduct chosen for analysis because of its prominent role in nucleic
acid chemistry and biology and the high frequency of O6-
alkylguanine lesions.8,9This adduct results naturally from exposure
to environmental carcinogens8a,band is highly mutagenic, causing
G to C and G to T transversion, and G to A transition mutations.8c,d
O6-Alkylguanine adducts have altered hydrogen-bonding capacity,
increased size, and decreased hydrophilicity relative to G (Figure 1).
On the basis of molecular modeling studies,10we anticipated that
a diaminonaphthyl-derived nucleoside (2, dNap; Figure 1) would
possess a hydrogen-bonding capacity complementary to O6-BnG
and favorable π-π stacking and hydrophobic interactions between
the benzyl moiety of O6-BnG and the naphthyl moiety of dNap 2.
To evaluate the O6-BnG:dNap base pair in duplex DNA, we
prepared a series of oligonucleotides containing selected combina-
tions of DNA adduct, synthetic nucleoside, and/or natural bases.
Nucleoside 2 was synthesized from diaminonaphthalene 3 (Scheme
1). Treatment of 3 with ethyl chloroformate produced perimidinone
4 (70% yield), which was coupled with bistoluoyl chloroglycoside
to yield the ?-isomer of 5 as the major product. Deprotection, 5′-
tritylation, and conversion to the 3′-phosphoramidite 6, required
for oligonucleotide synthesis, were achieved in 50% yield overall.
Duplex DNA stability was determined by thermal denaturation
of synthetic oligonucleotides. Melting temperatures (Tm) were mea-
sured for complementary sequences 5′-TTGTCGGTATAXC GG-
3′ and 5′-CCGYTATACCGACAA-3′ with varying bases incorpo-
rated at positions X and Y. The results indicate that O6-BnG:dNap
is markedly stable (8.0 µM) with a Tmvalue one degree lower than
that of the natural dG:dC pair (Figure 2, D1 Tm) 60.3 vs D6 Tm
) 59.3). Sequence D5 represents a situation in which natural DNA
is damaged, giving rise to the O6-BnG adduct and a diminished
thermal stability. Further, the adduct:probe pair Tmwas compared
to O6-BnG paired with canonical bases. These combinations have
diminished stabilities relative to the synthetic pair by 5.0, 6.6, 4.7,
and 5.9 °C for dG, dA, dC, and dT, respectively (Figure 2). Simi-
larly, for dNap paired opposite the natural bases, Tmdiminished to
55.3, 54.8, 52.5, and 52.5 °C for dG, dA, dC, and dT, respectively.
These data are comparable to optimized synthetic base pairs, in
which approximate ranges of 4-9 °C in Tm depressions are
considered highly stable and orthogonal systems.3eTmvalues for
point mutations in D1, which reflect the selectivity of natural base
pairs, decrease by an estimated average of 9 °C.11
Figure 1. Schematic representation of base-pair interactions for a standard
G:C pair, alkylation-damaged O6-BnG 1:C pair and proposed adduct:probe
combination (dR ) deoxyribose).
aReagents and conditions: (a) ethyl chloroformate, THF; (b) bistoluoyl
chloroglycoside, NaH/THF; (c) NaOMe/methanol; (d) 4,4′-dimethoxytrityl
chloride, pyridine; (e) N,N′-diisopropyl-2-O-cyanoethyl phosphoramidic
chloride, Et3N, CH2Cl2.
Figure 2. Thermal stabilities of natural, damaged, and dNap DNA.
Published on Web 04/03/2007
4882 9 J. AM. CHEM. SOC. 2007, 129, 4882-4883
10.1021/ja070688g CCC: $37.00 © 2007 American Chemical Society
A goal for potential biological applications is that dNap distin-
guish between isomeric adduct structures resulting from competing
positions of base alkylation. We compared damaged oligonucle-
otides that contained O6-BnG or the isomeric adduct N2-ben-
zyldeoxyguanosine (7, N2-BnG). The N2-BnG:dNap pair was less
stable, but the difference was small (Tmof 57.4 °C, 1.9 °C lower than
that of O6-BnG:dNap). Many known synthetic nucleosides form
stable self-pairs in duplex DNA.3e-gSimilarly, the Tmfor a duplex
containing dNap:dNap is 60.3 °C, essentially as stable as dG:dC.
Thermodynamic relationships for key base pairs were evaluated
further by a van’t Hoff analysis (Table 1).12The relative free-energy
changes parallel those observed for Tmvalues, with high Tmvalues
associated with high free-energy changes upon duplex formation.
Entropic contributions were similar for each example.
To verify whether the modified oligonucleotides formed strictly
duplex structures, a titration study (Job plot) was performed by
measuring UV absorbances at 260 nm for various molar ratios of
the single strands. These data (Figure S3-S5, Supporting Informa-
tion) clearly indicate 1:1 stoichiometric binding. To probe the
helicity of the key duplexes in Figure 2, circular dichroism (CD)
spectra of duplexes D1, D2, and D6 were obtained. The resulting
CD spectra (Figure S6, Supporting Information) display positive
signals at 271-274 nm and negative signals at 248-251 nm,
indicating a B-form conformation of DNA duplexes. Similar
patterns of CD spectra suggest that the synthetic base pair does
not significantly perturb duplex conformation.
The relative stereochemistry of free dNap was assigned on the
basis of NOESY correlations (obtained from 5, Supporting Informa-
tion) and confirmed by X-ray analysis of the free nucleoside 2
(Figure 3). Both indicate that the nucleoside favors a syn glycosidic
torsion angle, contrasting the anti-conformation proposed to maxi-
mize H-bonding and π-stacking interactions (Figure 1). The energy
barrier between syn and anti nucleoside conformations (Figure 1)
is typically low,13and there may be structural differences among
the same free nucleoside in solution or solid state, duplex DNA, or
in the presence of other nucleosides.14,15The relationship of the dNap
network in the crystal structure indicates a potential for π-π inter-
actions with an extensive array of face-to-face slipped π-π stacking
and hydrogen-bonding interactions of the naphthalene and deox-
yribose moieties, respectively, with adjacent dNaps arranged in
alternating orientations with about 3.2 Å between neighboring
parallel naphthyl groups. Further studies to determine the nucleoside
structure in the context of duplex DNA in the presence and absence
of adduct are required to understand the origin of the experimentally
observed stabilizing effect.
The novel naphthalene-derived nucleoside forms an orthogonal
and thermodynamically stable base pair with the biologically
significant DNA adduct O6-BnG. This is the first report of a stable
DNA base pair comprised of a biologically relevant bulky DNA
adduct and a designed nucleoside partner. Synthetic nucleosides
that base pair specifically with DNA adducts have diverse potential
utility in the study of the impacts of chemical modification on DNA
biology and chemistry. Continued studies are aimed at gaining a
detailed understanding of the physical and structural origin of
adduct:probe base-pair stability, the design of more selective
analogues, and applications as structural probes.
Acknowledgment. We acknowledge the NIH (CA108604) for
support. J.G. thanks the University of Minnesota Cancer Center
for a postdoctoral fellowship. We thank Dr. Besik Kankia for helpful
suggestions, and Dr. Yuk Sham, University of Minnesota Super-
computing Institute, for assistance with molecular models. Crystal-
lographic analysis was carried out by Dr. Victor G. Young, Jr. at
the X-ray Laboratory of the University of Minnesota.
Supporting Information Available: Syntheses, NMR data, thermal
denaturation studies, crystallographic analysis, Job plots, and CD
spectra. This material is available free of charge via the Internet at
(1) (a) Singer, B.; Runberger, D. Molecular Biology of Mutagens &
Carcinogens; Plenum Press: New York, 1983. (b) Phillips, D. H. The
Formation of DNA Adducts. In The Cancer Handbook; Alison, M. R.,
Ed.; Nature Publishing Group: London, 2002; pp 293-306.
(2) (a) Singer, B.; Essigmann, J. M. Carcinogenesis 1991, 12, 949-955. (b)
Loechler, E. L. Carcinogenesis 1996, 17, 895-902. (c) Hecht, S. S. Nat.
ReV. Cancer 2003, 3, 733-744.
(3) (a) Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature
1990, 343, 33-37. (b) Moran, S.; Ren, R. X. F.; Rumney, S.; Kool, E. T.
J. Am. Chem. Soc. 1997, 119, 2056-2057. (c) Kool, E. T. Acc. Chem.
Res. 2002, 35, 936-943. (d) Benner, S. A. Acc. Chem. Res. 2004, 37,
784-797. (e) Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz,
P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 7621-7632. (f)
Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg,
F. E. J. Am. Chem. Soc. 2005, 122, 3274-3287. (g) Henry, A. A.; Olsen,
A. G.; Matsuda, S.; Yu, C.; Geierstanger, B. H.; Romesberg, F. E. J. Am.
Chem. Soc. 2005, 126, 6923-6931.
(4) (a) Bloom, L. B.; Otto, M. R.; Beechem, J. M.; Goodman, M. F.
Biochemistry 1993, 32, 11247-11258. (b) Matray, T. J.; Kool, E. T.
Nature 1999, 399, 704-708. (c) Sun, L.; Xhang, K.; Zhou, L.; Hohler,
P.; Kool, E. T.; Yuan, G.; Wang, Z.; Taylor, J. S. Biochemistry 2003, 42,
9431-9437. (d) Zhang, X. M.; Lee, I.; Zhou, X.; Berdis, A. J. J. Am.
Chem. Soc. 2006, 128, 143-149. (e) Mizukami, S.; Kim, T. W.; Helquist,
S. A.; Kool, E. T. Biochemistry 2006, 45, 2772-2778.
(5) Gao, J.; Liu, H.; Kool, E. T. J. Am. Chem. Soc. 2004, 126, 11826-11831.
(6) (a) Hirao, I.; Ohtsuki, T.; Mitsui, T.; Yokoyama, S. J. Am. Chem. Soc.
2000, 122, 6118-6119. (b) Mitsui, T.; Kitamura, A.; Kimoto, M.; To,
T.; Sato, A.; Hirao, I.; Yokoyama, S. J. Am. Chem. Soc. 2003, 125, 5298-
5307. (c) Kimoto, M.; Endo, M.; Mitsui, T.; Okuni, T.; Hirao, I.;
Yokoyama, S. Chem. Biol. 2004, 11, 47-55.
(7) Hirao, I.; Kimoto, M.; Mitsui, T.; Fujiwara, T.; Kawai, R.; Sato, A.;
Harada, Y.; Yokoyama, S. Nat. Methods 2006, 3, 729-735.
(8) (a) Moschel, R. C.; Hudgins, W. R.; Dipple, A. J. Org. Chem. 1980, 45,
533-535. (b) Peterson, L. A. Chem. Res. Toxicol. 1997, 10, 19-26. (c)
Mitra, G.; Pauly, G. T.; Kumar, R.; Pei, G. K.; Hughes, S. H.; Moschel,
R. C.; Barbacid, M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8650-8654.
(d) Pauly, G. T.; Moschel, R. C. Chem. Res. Toxicol. 2001, 14, 894-
900. (e) Dolan, M. E.; Pegg, A. E. Clin. Cancer Res. 1997, 3, 837-847.
(9) (a) Mishina, Y.; Duguid, E. M.; He, C. Chem. ReV. 2006, 106, 215-236.
(b) Margison, G. P.; Santibanez, K.; Mauro, F.; Povey, A. C. Mutagenesis
2002, 17, 483-487.
(10) Using the program Insight II and information from adduct structures within
a ternary complex with a DNA polymerase, such as: Ling, H.; Sayer, J.
M.; Plosky, B. S.; Yagi, H.; Buodsocq, R.; Woodgate, R.; Jerina, D. M.;
Yang, W. Proc. Natl. Acad. Sci. U.S.A. 2004, 8, 2265-2269.
(11) Calculated for all point mutations at X,Y in Figure 2 sequence using the
DINAMelt server, http://www.bioinfo.rpi.edu.
(12) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620.
(13) Rosemeyer, H.; To ´th, G.; Golankiewicz, B.; Kazimierczuk, Z.; Bourgeois,
W.; Kretschmer, U.; Muth, H.; Seela, F. J. Org. Chem. 1990, 55, 5784-
(14) (a) Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem. 1998, 63,
9652-9656. (b) Guckian, K. M.; Krugh, T. R.; Kool, E. T. J. Am. Chem.
Soc. 2000, 122, 6841-6847.
(15) Haschemeyer, A. E. V.; Sobell, H. M. Acta Crystallogr. 1965, 19,
Table 1. Thermodynamic Parameters for Duplex Formation
Figure 3. Stacking interactions in 2 indicated in X-ray crystal structure
(unit cell dimensions: a ) 6.5 Å; b ) 8.7 Å; c ) 23.5 Å).
C O M M U N I C A T I O N S
J. AM. CHEM. SOC. 9 VOL. 129, NO. 16, 2007 4883