An emissive C analog distinguishes between G, 8-oxoG, and t

Article abstract of DOI:10.1021/ol802656n

A minimally disruptive fluorescent dC analog provides a rapid and non-destructive method for in vitro detection of G, 8-oxoG, and T, the downstream transverse mutation product.

Full text of DOI:10.1021/ol802656n

ORGANIC
LETTERS
2009
Vol. 11, No. 5
1115-1118
An Emissive C Analog Distinguishes
between G, 8-oxoG, and T
Nicholas J. Greco, Renatus W. Sinkeldam, and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California,
San Diego, 9500 Gilman DriVe, La Jolla, California 92093-0358
ytor@ucsd.edu
Received November 25, 2008
ABSTRACT
A minimally disruptive fluorescent dC analog provides a rapid and non-destructive method for in vitro detection of G, 8-oxoG, and T, the
downstream transverse mutation product.
One of the most thoroughly examined DNA base modifications
is 7,8-dihydro-8-oxoguanine (8-oxoG), a mutagenic product of
oxidative damage by reactive oxygen species.1 The presence
of 8-oxoG is frequently viewed as a marker for cellular oxidative
stress, a condition that has been linked to carcinogenesis.2 The
significance of this seemingly minor base damage results from
8-oxoG’s ability to deceive DNA polymerases and form a stable
(syn)8-oxoG·A base pair by presenting its Hoogsteen face,
thereby mimicking T (Figure 1).3 Unless repaired, this might
cause G to T transversion mutations during DNA synthesis
(Figure 1).4 Not surprisingly, base-excision repair mechanisms
have evolved to correct such deleterious base modification
products.5
Figure 1. Base pairing along the DNA damage pathway from G·C
to T·A (its transverse mutation product) via 8-oxoG.
A non-destructive and real-time fluorescence-based detection
of 8-oxoG, its repair, and induced mutation processes in
oligonucleotides could significantly advance the in vitro bio-
chemical evaluation of this important DNA lesion.6It would
complement existing procedures that rely on chromatographic,
electrophoretic and immunological methods.7,8 Toward this
end, we hypothesized that one could take advantage of the
distinct redox properties of 8-oxoG.9 Electrochemical mea-
(1) (a) Beckman, K. B.; Ames, B. N. J. Biol. Chem. 1997, 272, 19633–
19636. (b) Burrows, C. M.; Muller, J. Chem. ReV. 1998, 98, 1109–1152.
(c) Bjelland, S.; Seeberg, E. M. Mutat. Res. 2003, 531, 37–80. (d) Cadet,
J.; Douki, T.; Gasparutto, D.; Ravanat, J.-L. Mutat. Res. 2003, 531, 5–23.
(e) Greenberg, M. M. Biochem. Soc. Trans. 2004, 32, 46–50. (f) Neeley,
W. L.; Essigmann, J. M. Chem. Res. Toxicol. 2006, 19, 491–505. (g) Cooke,
M. S.; Evans, M. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 13535–13536.
(2) (a) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 7915–7922. (b) Loft, S.; Poulsen, H. E. J. Mol. Med.
1996, 74, 297–312. (c) Wiseman, H.; Halliwell, B. Biochem. J. 1996, 313,
17–29. (d) Klaunig, J. E.; Kamendulis, L. M. Annu. ReV. Pharmacol.
Toxicol. 2004, 44, 239–267. (e) Hirano, T. J. Radiat. Res. 2008, 49, 329–
340.
(4) Wood, M. L.; Dizdaroglu, M.; Gajewski, E.; Essigmann, J. M.
Biochemistry 1990, 29, 7024–7032.
(5) For recent reviews, see: (a) Fromme, J. C.; Verdine, G. L. AdV.
Protein Chem. 2004, 69, 1–41. (b) David, S. S.; O’Shea, V. L.; Kundu, S.
Nature 2007, 447, 941–950.
(6) 8-oxoG’s low cellular abundance, complex localization, and chemical
degradation pathways make the development of small probes for its
intracellular detection a formidable task.
(3) Leonard, G. A.; Guy, A.; Brown, T.; Teoule, R.; Hunter, W. N.
Biochemistry 1992, 31, 8415–8420.
10.1021/ol802656n CCC: $40.75
Published on Web 02/05/2009
2009 American Chemical Society

surements show that 8-oxoG is more easily oxidized
compared to (E1/2 0.75 and 1.3 vs NHE,
Useful uridine-based nucleosides, fulfilling these criteria, were
obtained by conjugating five-membered aromatic heterocycles
such as furan at the 5 position (e.g., 2).15-18 Among the various
heterocycles conjugated to dU, the furan moiety was found to
yield the most favorable photophysical characteristics.15,17a We
therefore anticipated the analogous furan-modified cytosine
nucleobase to be emissive and responsive.
The 5-modified nucleosides are easily obtained using a
coupling reaction between the 5-iodo substituted dU (1) and
the corresponding stannylated heterocycles (Scheme 1). Con-
G
≈
respectively).9a,10 Consequently, chemical approaches relying
on 8-oxoG’s susceptibility to oxidation and covalent trapping
of the oxidized products in duplex DNA have been re-
ported.11 Since fluorescence quenching frequently occurs via
photoinduced electron transfer (PET) mechanisms,12 we
suspected that 8-oxoG is likely to be a more effective
quencher of certain fluorophores compared to G, its precur-
sor.13 Here we describe the design, synthesis, photophysical
evaluation, incorporation, and implementation of a simple
isomorphic fluorescent dC analog 5 that, upon incorporation
into an oligonucleotide, photophysically distinguishes be-
tween 8-oxoG and G on the complementary strand. Not only
is the damaged 8-oxoG-containing duplex highly quenched
and the “repaired” G-containing duplex more emissive, the
transverse mutated duplex containing T instead of G displays
the most intense emission. This furan-containing emissive
nucleobase therefore provides signature emission profiles for
all key nucleobases involved in this DNA damage pathway
(Figure 1).14
Scheme 1
.
Synthesis of Furan dC Analog 5 and Its
Corresponding Amidite
The primary design principle for minimally perturbing
emissive nucleobases dictates maintaining the highest possible
structural similarity to the natural nucleobases, while signifi-
cantly improving their photophysical properties.15,16 Specifi-
cally, an isolated absorption band for selective excitation,
enhanced quantum yield over the native nucleobases, and
sensitivity to changes in the microenvironment are desired.
(7) (a) Shigenaga, M. K.; Ames, B. A. Free Radical Biol. Med. 1991,
10, 211–216. (b) Muller, J. G.; Duarte, V.; Hickerson, R. P.; Burrows, C. J.
Nucleic Acids Res. 1998, 26, 2247–2249. (c) Ropp, P. A.; Thorp, H. H.
Chem. Biol. 1999, 6, 599–605. (d) Pouget, J.-P.; Douki, T.; Richard, M.-J.;
Cadet, J. Chem. Res. Toxicol. 2000, 13, 541–549. (e) Soultanakis, R. P.;
Melamede, R. J.; Bespalov, I. A.; Wallace, S. S.; Beckman, K. B.; Ames,
B. N.; Taatjes, D. J.; Janssen-Heininger, Y. M. Free Radical Biol. Med.
2000, 28, 987–998. (f) Persinger, R. L.; Melamede, R.; Bespalov, I.;
Wallace, S.; Taatjes, D. J.; Janssen-Heininger, Y. Exp. Gerontol. 2001, 36,
1483–1494. (g) Gore, M. R.; Szalai, V. A.; Ropp, P. A.; Yang, I. V.;
Silverman, J. S.; Thorp, H. H. Anal. Chem. 2003, 75, 6586–6592
(8) For fluorescence-based kinetic analysis of 8-oxoG DNA glycosylase;
see: Kuznetsov, N. A.; Koval, V. V.; Nevinsky, G. A.; Douglas, K. T.;
Zharkov, D. O.; Fedorova, O. S. J. Biol. Chem. 2007, 282, 1029–1038
(9) (a) Oliveira Brett, A. M.; Piedade, J. A. P.; Serrano, S. H. P.
.
version of the acetate-protected furan-modified dU analog 3 to
the desired dC analog 5 is accomplished by activation of the 4
position as an aryl sulfonate ester followed by a displacement
reaction with ammonia,19 providing the fully deprotected furan-
modified dC analog 5 (Scheme 1).17a,20 Silyl protection of the
furan-modified dU 4 facilitated the conversion to the dC
analog 6 with retention of hydroxyl protection, thus allowing
for standard benzamide protection of the exocyclic amine
to give 7. Desilylation and protection of the 5′-hydroxyl as
the 4,4′-dimethoxytrityl derivative (9) followed by phosphi-
tylation of the unprotected 3′-hydroxyl afforded 10, the
building block necessary for automated DNA synthesis
(Scheme 1).20
.
Electroanalysis 2000, 12, 969–973. (b) Diculescu, V. C.; Chiorcea Paquim,
A.-M.; Oliveira Brett, A.-M. Sensors 2005, 5, 377–393
.
(10) (a) Berger, M.; Anselmino, C.; Mouret, J.-F.; Cadet, J. J. Liq.
Chromatogr. 1990, 13, 929–940. (b) Goyal, R. N.; Dryhurst, G. J.
Electroanal. Chem. 1992, 135, 75–91. (c) Yanagawa, H.; Ogawa, Y.; Ueno,
M. J. Biol. Chem. 1992, 267, 13320–13326
.
(11) (a) Xue, L.; Greenberg, M. M. J. Am. Chem. Soc. 2007, 129, 7010–
7011. (b) Xue, L.; Greenberg, M. M. Angew. Chem., Int. Ed. 2007, 46,
561–564.
(12) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.; Kamagata,
Y.; Kanagawa, T.; Kurane, R. Anal. Sci. 2001, 17, 155–160.
(13) A modified G-clamp was reported to be quenched by 8-oxoG, but
detecting the damaged base was limited to nucleosides in chloroform and
micellar solutions. See: Nakagawa, O.; Ono, S.; Li, Z.; Tsujimoto, A.;
Sasaki, S. Angew. Chem., Int. Ed. 2007, 46, 4500–4503.
The absorption spectrum of an aqueous solution of 5 shows,
in addition to the typical high energy band seen in the parent
(14) For other emissive C analogs, see: (a) Godde, F.; Toulme, J. J.;
Moreau, S. Nucleic Acids Res. 2000, 28, 2977–2985. (b) Wilhelmsson,
L. M.; Holme´n, A.; Lincoln, P.; Nielsen, P. E.; Norde´n, B. J. Am. Chem.
Soc. 2001, 123, 2434–2435. (c) Liu, C. H.; Martin, C. T. J. Biol. Chem.
2002, 277, 2725–2731. (d) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem.
Soc. 2003, 125, 4972–4973. (e) Berry, D. A.; Jung, K. Y.; Wise, D. S.;
Sercel, A. D.; Pearson, W. H.; Mackie, H.; Randolph, J. B.; Somers, R. L.
Tetrahedron Lett. 2004, 45, 2457–2461. (f) Engman, K. C.; Sandin, P.;
Osborne, S.; Brown, T.; Billeter, M.; Lincoln, P.; Norde´n, B.; Albinsson,
B.; Wilhelmsson, L. M. Nucleic Acids Res. 2004, 32, 5087–5095. (g)
Tinsley, R. A.; Walter, N. G. RNA 2006, 12, 522–529. (h) Marti, A. A.;
Jockusch, S.; Li, Z. M.; Ju, J. Y.; Turro, N. J. Nucleic Acids Res. 2006, 34,
e50. (i) Wojciechowski, F.; Hudson, R. H. E. J. Am. Chem. Soc. 2008,
130, 12574–12575.
(15) Greco, N. J.; Tor, Y. J. Am. Chem. Soc. 2005, 127, 10784–10785.
Greco, N. J.; Tor, Y. Nat. Protoc. 2007, 2, 305–316.
(16) Srivatsan, S. G.; Greco, N. J.; Tor, Y. Angew. Chem., Int. Ed. 2008,
47, 6661–6665.
(17) (a) Greco, N. J.; Tor, Y. Tetrahedron 2007, 63, 3515–3527. (b)
Tor, Y.; Del Valle, S.; Jaramillo, D.; Srivatsan, S. G.; Rios, A.; Weizman,
H. Tetrahedron 2007, 63, 3608–3614.
(18) (a) Srivatsan, S. G.; Tor, Y. J. Am. Chem. Soc. 2007, 129, 2044–
2053. Srivatsan, S. G.; Tor, Y. Tetrahedron 2007, 63, 3601–3607. (b)
Srivatsan, S. G.; Tor, Y. Nat. Protoc. 2007, 2, 1547–1555.
(19) Li, S.-N.; Piccirilli, J. A. J. Org. Chem. 2004, 69, 4751–4759.
(20) See Supporting Information for experimental details.
1116
Org. Lett., Vol. 11, No. 5, 2009

Figure 2. Absorption (dashed) and emission (solid) spectra of 5 in
water (blue), methanol (green), dichloromethane (orange), and
Figure 4. Steady-state Stern-Volmer plot for the titration of 5 with
dioxane (red) at 2.4 × 10-5 M.
8-oxo-2′-deoxyguanosine (stars, black dashed line), dGMP (X), and
TMP (open circles). Some data and error bars were omitted for
clarity (Figure S3.1 and Table S3.1, Supporting Information).20
nucleoside, a clear shoulder at ∼310 nm (Figure 2). Excitation
at this wavelength yields an emission profile with a maximum
at 443 nm, which tails deeply into the visible range, with a
relative quantum yield of 0.02 (Table 1).20,21 Lowering solvent
mally impacted the emission of 5 (Ksv ) 0.004 mM-1), 8-oxoG
was found to be a very effective quencher, even at low
concentrations (Ksv ) 16.5 mM-1).20
To investigate the potential of the emissive nucleoside to
photophysically discriminate between G, 8-oxoG, and T, an
oligonucleotide that contains 5 at a central position was
synthesized using 10 and standard solid-phase synthesis pro-
tocols (Figure 5).20 All modified oligonucleotides were char-
Table 1. Photophysical Properties of Nucleoside 5 in Various
Solvents20
solvent
λabs (nm) λem (nm)
Φ
I normalized
water
methanol
dichloromethane
dioxane
310
305
309
309
443
439
439
421
0.020
0.011
0.009
0.006
1.00
0.70
0.54
0.33
polarity results in a better defined long wavelength absorption
band (λmax ) 309 nm) and a hypsochromic shift in emission
maxima (λem ) 421 nm), which is associated with a
hypochromic effect (IWater/Dioxane ) 3) (Figure 2, Table 1).
Emission spectra of 5 in dioxane-water mixtures provide a
more detailed view of the hypsochromic shift that arises upon
decreasing the polarity of the chromophore’s microenvironment
(Figure S2.1, Supporting Information).20 Plotting the corrected
emission energy maximum of 5 versus the microenvironment
polarity described by ET(30) values22 results in a linear
correlation (Figure 3).23 Stern-Volmer titrations were con-
ducted to determine the differences between G and 8-oxoG’s
quenching abilities (Figure 4). Rewardingly, whereas G mini-
Figure 5. Oligonucleotide sequences where Y ) 8-oxoG.
acterized using MALDI TOF MS.20 Since modified C residues
can, in principle, deaminate to yield the corresponding U
derivatives, we unequivocally verified the presence of 5 and
absence of 2 in the modified oligonucleotide 11. Enzymatic
digestion reactions, followed by HPLC analysis against all
authentic nucleosides verified the presence of intact 5 and
absence of 2 in the modified oligonucleotide 11 (Figure S6.1
and Table S6.1, Supporting Information).20
Oligonucleotide 11, containing the furan-functionalized dC
5 was hybridized to three complementary oligonucleotides that
contain either G (oligo 12), 8-oxoG (oligo 13), or T (oligo 14)
opposite the emissive nucleotide (Figure 5). Thermal denatur-
ation (Table 2) and CD studies show no appreciable difference
between the modified (11·12, 11·13, and 11·14) and unmodified
duplexes (15·12, 15·13, and 15·14, respectively), suggesting the
small furan modification does not disrupt duplex formation,
stability, or structure (Figures S8.1 and S9.1, Supporting
Information).20
The emission spectra of the furan dC containing duplexes,
in both low (100 mM) and elevated (500 mM) ionic strength,
(21) Note that, although relatively low, this is easily detected by standard
benchtop fluorimeters with excellent signal-to-noise.
Figure 3. Correlating emission wavelengths with microscopic
polarity ET(30)22 for nucleoside 5 (filled circles and green line)20
and interpolation of corrected emission maxima23 of duplexes
containing 5 (arrows).
(22) Reichardt, C. Chem. ReV. 1994, 94, 2319–2358.
(23) Fluorescence spectra were corrected according to I(ν) ) λ2I(λ).
See Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic/Plenum: New York, 1999.
Org. Lett., Vol. 11, No. 5, 2009
1117

Thermal denaturation data illustrates that the dC/T containing
duplex 15·14 (as well as the analogous modified duplex 11·14)
are the least stable (Table 2). This is consistent with previous
observations illustrating this particular dual pyrimidine mismatch
to be rather unfavorable,24 therefore suggesting a local structural
perturbation. Fluorescence experiments carried out at elevated
ionic strengths (500 mM) for all duplexes resulted in similar
observations (Figure 6), rendering the potential higher abun-
dance of the more emissive single strand due to poor hybridiza-
tion unlikely.20
Further support for the proposed extrahelical residency of
the fluorescent nucleobase is obtained by interpolating the
emission energy of modified duplexes containing 5 and
environmental polarity using the linear correlation shown above
(Figure 3, Table 3). The emission spectra of the furanyl modified
Table 2. Thermal Denaturation of Control and Modified
Oligonucleotides
b
b
Tm (∆Tm
)
Tm (∆Tm )
Duplexa
(°C) 100 mM NaCl
(°C) 500 mM NaCl
15·12
15·13
15·14
11·12
11·13
11·14
51.8
47.7
34.5
51.5 (-0.3)
47.1 (-0.6)
34.7 (+0.2)
59.2
55.2
41.2
59.0 (-0.2)
54.5 (-0.7)
42.0 (+0.8)
a 1.0 × 10-6 M duplex DNA aqueous buffer pH ) 7.0. b ∆Tm ) modified
- unmodified.
are shown in Figure 6.23 While the perfect duplex 11·12, where
G is placed opposite 5, is significantly emissive, placing 5
opposite 8-oxoG (11·13) leads to considerable emission quench-
Table 3. Emission Maxima of Modified Duplexes in Phosphate
Buffers at Different NaCl Concentrations
λem/nm (cm-1
duplex 100 mM NaCl
)
intensity at λem/nm (cm-1
)
intensity at
λem/au
500 mM NaCl
λem/au
11·12
11·13
11·14
439 (22,779)
435 (22,989)
446 (22,422)
1.00
0.52
2.05
440 (22,727)
437 (22,883)
447 (22,371)
1.00
0.55
2.15
duplexes 11·12 and 11·13, where furanyl dC 5 can pair in a
Watson-Crick fashion, display emission maximum that cor-
relate to relatively apolar environments (Figures 3 and 6, Table
3), while duplex 11·14, where furanyl dC 5 is unable to pair in
a W-C fashion with T, shows a red-shifted emission maximum
that correlates to a polar environment, in agreement with an
extrahelical disposition.
In summary, we have shown that an isomorphic fluorescent
nucleoside 5 is a valuable probe for the detection of G, 8-oxoG,
and its transverse mutation product T by eliciting markedly
different emission intensities in conjunction with changes in
emission maxima. The effective synthesis and incorporation of
5 could facilitate rapid and non-destructive real-time fluorescence-
based methods for the in vitro monitoring of this DNA damage
pathway.25
Figure 6. Steady-state emission spectra of oligonucleotides 11·12
(5·G, blue), 11·13 (5·8-oxoG, red), and 11·14 (5·T, orange) at 5.0
× 10-6 M in 1.0 × 10-2 M phosphate aqueous buffer pH ) 7.0
containing 1.0 × 10-1 M NaCl (solid) and at 4.6 × 10-6 M in 1.0
× 10-2 M phosphate aqueous buffer pH ) 7.0 containing 5.0 ×
10-1 M NaCl (dashed).20
ing (ca. 2-fold), as hypothesized. Nucleoside 5 therefore clearly
distinguishes between G and 8-oxoG, its oxidized product. In
contrast, for duplex 11·14, where 5 is placed opposite T, a
substantial emission enhancement (ca. 4-fold compared to 11·13)
is observed (Figure 6, Table 3). Nucleoside 5 thus reports the
presence of T, the ultimate transversion mutation product
resulting from G oxidation to 8-oxoG, via enhanced emission.
These observations, illustrating signature emission profiles for
duplexes containing G, 8-oxoG, and T, suggest that the furan-
modified dC nucleoside 5 could be utilized to follow, in vitro,
DNA damage and its repair via this pathway using such
emissive oligonucleotides.
Acknowledgment. We thank the National Institutes of
Health (GM069773) for support.
Supporting Information Available: Experimental proce-
dures and biophysical data. This material is available free of
charge via the Internet at http://pubs.acs.org.
The observed fluorescence quenching of 5 by 8-oxoG can
be understood by the lower redox potential of 8-oxoG and
concomitant higher potency as an excited-state quencher
compared to G (Figure 4). Several observations collectively
suggest that the substantial fluorescence enhancement observed
for the 5·T mismatch in 11·14 is due to exposure of the emissive
nucleoside to a more polar environment, likely extrahelical.
OL802656N
(24) (a) Aboul-ela, F.; Koh, D.; Tinoco, I., Jr. Nucleic Acids Res. 1985,
13, 4811–4824. (b) Allawi, H. T.; SantaLucia, J., Jr. Nucleic Acids Res.
1998, 26, 2694–2701. (c) Hobza, P.; Sponer, J. Chem. ReV. 1999, 99, 3247–
3276.
(25) To globally follow this pathway, an independent method, estimating
the relative damaged/mutant population, might be needed.
1118
Org. Lett., Vol. 11, No. 5, 2009