A synthetic nucleoside probe that discerns a DNA adduct from unmodified DNA

Article abstract of DOI:10.1021/ja070688g

Selective pairing of engineering nucleosides in DNA duplexes provides a potential means to probe structurally modified DNA bases (i.e., DNA adducts) and address challenges associated with correlating adduct chemical structure with biological impact. The c

Full text of DOI:10.1021/ja070688g

Published on Web 04/03/2007
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: sturl002@umn.edu
Biologically reactive chemicals alkylate DNA and induce
1
structural modifications in the form of covalent adducts. Certain
bulky DNA adducts can persist, escape repair, and serve as
templates for polymerase-mediated DNA synthesis, resulting in
2
mutation and cancer. Correlating 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.
Figure 1. Schematic representation of base-pair interactions for a standard
G:C pair, alkylation-damaged O -BnG 1:C pair and proposed adduct:probe
combination (dR ) deoxyribose).
6
Dozens of thermodynamically stable synthetic base pairs have been
reported and continue to emerge as powerful tools in areas such as
Scheme 1 a
3
4
5
polymerase fidelity, DNA helix stability, nucleic acids with novel
6
3
functionality, and expanded genetic systems, to cite selected exam-
ples. Recently, Hirao and co-workers successfully have amplified
an entirely synthetic base pair. Amplified in a polymerase-mediated
7
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.
a
Reagents 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.
6
6
O -Benzyldeoxyguanosine (1, O -BnG; Figure 1) is a bulky DNA
adduct chosen for analysis because of its prominent role in nucleic
6
acid chemistry and biology and the high frequency of O -
alkylguanine lesions.8 This adduct results naturally from exposure
,9
to environmental carcinogens8 and is highly mutagenic, causing
a,b
8
c,d
G to C and G to T transversion, and G to A transition mutations.
6
O -Alkylguanine adducts have altered hydrogen-bonding capacity,
increased size, and decreased hydrophilicity relative to G (Figure 1).
On the basis of molecular modeling studies,10 we anticipated that
a diaminonaphthyl-derived nucleoside (2, dNap; Figure 1) would
6
possess a hydrogen-bonding capacity complementary to O -BnG
and favorable π-π stacking and hydrophobic interactions between
6
the benzyl moiety of O -BnG and the naphthyl moiety of dNap 2.
To evaluate the O -BnG:dNap base pair in duplex DNA, we
6
Figure 2. Thermal stabilities of natural, damaged, and dNap DNA.
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
that of the natural dG:dC pair (Figure 2, D1 T ) 60.3 vs D6 Tm
m
) 59.3). Sequence D5 represents a situation in which natural DNA
6
is damaged, giving rise to the O -BnG adduct and a diminished
1
4
). Treatment of 3 with ethyl chloroformate produced perimidinone
(70% yield), which was coupled with bistoluoyl chloroglycoside
thermal stability. Further, the adduct:probe pair T was compared
to O -BnG paired with canonical bases. These combinations have
m
6
to yield the â-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
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, T diminished to
m
55.3, 54.8, 52.5, and 52.5 °C for dG, dA, dC, and dT, respectively.
of synthetic oligonucleotides. Melting temperatures (T
sured for complementary sequences 5′-TTGTCGGTATAXC GG-
′ and 5′-CCGYTATACCGACAA-3′ with varying bases incorpo-
m
) were mea-
These data are comparable to optimized synthetic base pairs, in
which approximate ranges of 4-9 °C in T
m
depressions are
considered highly stable and orthogonal systems.3
e
3
m
T values for
6
rated at positions X and Y. The results indicate that O -BnG:dNap
is markedly stable (8.0 µM) with a T value one degree lower than
point mutations in D1, which reflect the selectivity of natural base
pairs, decrease by an estimated average of 9 °C.11
m
4882
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J. AM. CHEM. SOC. 2007, 129, 4882-4883
10.1021/ja070688g CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
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Table 1. Thermodynamic Parameters for Duplex Formation
significant DNA adduct O -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.
duplex
∆
H (kcal/mol)
∆
S (cal/K
‚
mol)
∆G
298K (kcal/mol)
∆∆G
298K (kcal/mol)
D1
D2
D6
-85.9
-67.2
-79.2
-232
-195
-218
-17.7
-9.1
-14.2
8.6
3.5
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.
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).
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-
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
http://pubs.acs.org.
6
2
otides that contained O -BnG or the isomeric adduct N -ben-
2
2
zyldeoxyguanosine (7, N -BnG). The N -BnG:dNap pair was less
stable, but the difference was small (T of 57.4 °C, 1.9 °C lower than
m
References
6
that of O -BnG:dNap). Many known synthetic nucleosides form
stable self-pairs in duplex DNA.3
e-g
(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.
m
Similarly, the T for 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).12 The relative free-energy
changes parallel those observed for T values, with high T values
m m
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.
(
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
1
990, 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,
(
9
431-9437. (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.
2
000, 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.
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
(
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,
(Figure 3). Both indicate that the nucleoside favors a syn glycosidic
5
33-535. (b) Peterson, L. A. Chem. Res. Toxicol. 1997, 10, 19-26. (c)
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,13 and there may be structural differences among
the same free nucleoside in solution or solid state, duplex DNA, or
in the presence of other nucleosides.14,15 The 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.
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-
9
00. (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
002, 17, 483-487.
(
2
(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.; T o´ th, G.; Golankiewicz, B.; Kazimierczuk, Z.; Bourgeois,
W.; Kretschmer, U.; Muth, H.; Seela, F. J. Org. Chem. 1990, 55, 5784-
5790.
(14) (a) Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem. 1998, 63,
9
652-9656. (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,
125-130.
The novel naphthalene-derived nucleoside forms an orthogonal
and thermodynamically stable base pair with the biologically
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