Photoswitchable formation of a DNA interstrand cross-link by a coumarin-modified nucleotide

Article abstract of DOI:10.1002/anie.201310609

A coumarin-modified pyrimidine nucleoside (1) has been synthesized using a Cu^I-catalyzed click reaction and incorporated into oligodeoxynucleotides (ODNs). Interstrand cross-links are produced upon irradiation of ODNs containing 1 at 350 nm. Cross-linking occurs through a [2+2] cycloaddition reaction with the opposing thymidine, 2'-deoxycytidine, or 2'-deoxyadenosine. A much higher reactivity was observed with dT than dC or dA. Irradiation of the dT-1 and dC-1 cross-linked products at 254 nm leads to a reversible ring-opening reaction, while such phenomena were not observed with dA-1 adducts. The reversible reaction is ultrafast and complete within 50-90 s. Consistent photoswitching behavior was observed over 6 cycles of irradiation at 350 nm and 254 nm. To the best of our knowledge, this is the first example of photoswitchable interstrand cross-linking formation induced by a modified pyrimidine nucleoside. A [2+2] cycloaddition reaction between a coumarin-modified pyrimidine nucleoside and the opposing thymidine, 2'-deoxycytidine, or 2'-deoxyadenosine on a DNA strand generates photoswitchable interstrand cross-links (ICLs) in the DNA.

Full text of DOI:10.1002/anie.201310609

Angewandte
Chemie
DOI: 10.1002/anie.201310609
DNA Cross-Linking
Photoswitchable Formation of a DNA Interstrand Cross-Link by
a Coumarin-Modified Nucleotide**
Mohammad Mojibul Haque, Huabing Sun, Shuo Liu, Yinsheng Wang, and Xiaohua Peng*
Abstract: A coumarin-modified pyrimidine nucleoside (1) has
been synthesized using a CuI-catalyzed click reaction and
incorporated into oligodeoxynucleotides (ODNs). Interstrand
cross-links are produced upon irradiation of ODNs containing
1 at 350 nm. Cross-linking occurs through a [2+2] cyclo-
addition reaction with the opposing thymidine, 2’-deoxycyti-
dine, or 2’-deoxyadenosine. A much higher reactivity was
observed with dT than dC or dA. Irradiation of the dT-1 and
dC-1 cross-linked products at 254 nm leads to a reversible ring-
opening reaction, while such phenomena were not observed
with dA-1 adducts. The reversible reaction is ultrafast and
complete within 50–90 s. Consistent photoswitching behavior
was observed over 6 cycles of irradiation at 350 nm and
254 nm. To the best of our knowledge, this is the first example
of photoswitchable interstrand cross-linking formation
induced by a modified pyrimidine nucleoside.
a non-invasive method with high spatio-temporal resolution
and control and offers the options of orthogonality. Recently,
the research groups of Freccero and Zhou reported the
photoinducible formation of quinone methide from biphenyl
or binol quaternary ammonium salts[13–15] and formation of
vinylidene-quinone methides from 2-alkynylphenols.[16] Qui-
none methides are highly reactive electrophiles which effi-
ciently cross-link DNA double strands.[11,13–15] Greenberg and
co-workers[17–21] described several modified nucleosides that
efficiently induced the interstrand cross-linking of DNA upon
photoirradiation. Recently, we reported hypoxia-selective
ICL formation from nitroimidazole-modified thymidine
upon UV irradiation.[22]
Photoactive molecules have also been studied for the
construction of DNA-based reversible photoswitches and
photo-manipulation of DNA.[23–28] For example, psoralens,
a class of naturally occurring photoreactive products, are
capable of cross-linking duplex and triplex DNA.[25–28] They
have been used as probes of nucleic acid structure and
function, for therapeutic gene modulation, and for studying
DNA damage and repair.[26–28] Coumarins show many advan-
tages such as high fluorescence quantum yield, large Stokes
shift, excellent photostability, and low toxicity. Coumarin
derivatives have been widely used in the fields of biology,
medicine, cosmetics, and as fluorescent chemosensors for
DNA, RNA, and protein detection.[29,30] However, there is no
report on the photoactivity of coumarin with DNA. Here, we
report the first coumarin-modified nucleoside that induces
photoswitchable formation of DNA ICLs upon UV irradi-
ation. A coumarin-modified thymidine efficiently induces
ICL formation upon photoirradiation at 350 nm, while the
cross-linking can be reversed by irradiation at 254 nm. The
cross-linking site was determined by LC-MS/MS as well as by
cleavage of gel-purified cross-linked DNA with hydroxyl
radicals.
A coumarin moiety has been conjugated to thymidine by
a Cu-catalyzed azide-alkyne cycloaddition reaction using
azide-modified thymidine 2 and an alkyne-modified coumarin
3.[18,31] Compound 1 was converted into its phosphoramidite
building block 5 under standard conditions (Scheme 1).
Oligodeoxynucleotides (ODNs) containing 1 were synthe-
sized by automated solid-phase synthesis using 5 and con-
firmed by MALDI-TOF-MS analysis (see Table S1 in the
Supporting Information).
The DNA duplex was photoirradiated at 350 nm for
50 min using a Rayonet Photochemical Chamber Reactor
(Model RPR-100). A wavelength of 350 nm was chosen
because near-UV light (> 300 nm) is compatible with living
cells and is almost not absorbed by most biological molecules
other than the coumarin-modified ODNs. Moreover, the
D
NA interstrand cross-links (ICLs) covalently link two
DNA strands, which can block DNA replication, transcrip-
tion, and any other processes requiring strand separation;
thus, they have a strong impact on the biological function of
nucleic acids. Chemical agents capable of inducing ICLs have
been developed for biological applications, such as for DNA
damage and repair studies,[1–5] as anticancer agents,[6] for
nucleic acid detection,[7,8] for targeting telomeric G-quadru-
plex structures,[9,10] and for the construction of DNA nano-
materials.[11] Over the past few decades, several research
groups have developed novel chemical methods for inducing
ICL formation, such as photoirradiation, oxidation, reduc-
tion, fluoride induction, and H2O2 induction.[12] Among these
methods, light induction is particularly important as it is
[*] M. M. Haque, H. Sun, Prof. X. Peng
Department of Chemistry and Biochemistry
University of Wisconsin Milwaukee
3210 N. Cramer St., Milwaukee, WI 53211 (USA)
E-mail: pengx@uwm.edu
S. Liu, Prof. Y. Wang
Department of Chemistry
University of California Riverside
332 Chemical Sciences Building, 501 Big Springs Road
Riverside, CA 92521-0403 (USA)
[**] We are grateful for financial support from the National Cancer
Institute (1R15CA152914-01), UWM Research Growth Initiative
(RGI101X234), WiSys technology foundation (applied research
grant award), and the Great Milwaukee Foundation (Shaw Scientist
Award; to X.P.) and the National Institute of Environmental Health
Sciences (R01 ES019873; to Y.W.). Support from the distinguished
graduate student fellowship for H.S. is also acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310609.
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of 350 nm. The UV irradiation of duplex 7
resulted in a new band whose migration is
severely retarded relative to unreacted
oligonucleotide, indicative of the formation
of interstrand cross-linked material, while
this was not observed with native DNA
duplex 6 (Figure 1). The generation of ICLs
induced by 1 followed first-order kinetics
with a rate constant (kICL) of 9.24 ꢀ 0.25 ꢀ
10ꢁ4 sꢁ1 (see Figure S1 in the Supporting
Information). The cross-linked product was
isolated and characterized by mass spec-
trometry and fluorescence measurements.
ODN 7a showed strong fluorescence with
an emission maximum at 392 nm. However,
a greatly decreased fluorescence intensity
was observed with the cross-linked product
8 (8a—c, Figure 2). We propose that the
cross-link formation may lead to the
destruction of the conjugation system of
the coumarin moiety.
Scheme 1. Synthesis of compound 1 and its phosphoramidite building block 5. Reagents:
a) Cu2SO4 and sodium ascorbate; b) dimethoxytrityl chloride (DMTCl), pyridine; c) 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine and CH2Cl2.
coumarin moiety can be excited to singlet and triplet
excitation states upon irradiation with light at a wavelength
Figure 2. Fluorescence emission spectra of coumarin-modified ODN
7a, ICLs (8a–c) induced by coumarin after photoirradiation, and the
unmodified ODN 6a. The reaction mixture contained 5.0 mm DNA,
100 mm NaCl, and 10 mm phosphate buffer (pH 7.0).
Initially, the hydroxyl radical footprinting of gel-purified
cross-linked DNA was used to determine which nucleotides
were covalently bonded with one another.[17] Each ODN was
either 5’- or 3’-32P-labeled. As expected, cross-linking oc-
curred mainly at the position of 1 (see Figure S2 in the
Supporting Information). However, the exact cross-linking
sites in the complementary strand could not be determined by
the cleavage reaction with hydroxyl radicals, possibly because
of the presence of multiple reaction sites. To exploit this
further, we used LC-MS and MS/MS to determine the
identities of the nucleobase(s) in the opposite strand that is
(are) cross-linked with the coumarin-modified thymidine
moiety. To this end, we digested the isolated ICL products
obtained from duplexes 7 and 9 with a cocktail of four
enzymes to release the ICL as a dinucleoside remnant, by
following previously published procedures,[32] and subjected
Figure 1. ICL formation upon UV irradiation at 350 nm for 50 min.
Phosphorimage autoradiogram of 20% denaturing PAGE analysis of
the ICL products: lane 1, unmodified duplex 6; lanes 2–5, duplexes 7
and 9–11 containing coumarin-modified ODNs hybridized with oppos-
ing sequences containing A, C, T, and G; lanes 6–9, duplexes 12–15
containing coumarin-modified ODNs hybridized with opposing
sequences containing only an A, T, G, and C in opposite sequences.
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the resulting mixture to LC-MS and MS/MS analyses. Our
LC-MS/MS results revealed, in the digestion mixture, the
presence of dinucleosides, with the coumarin-derived thymi-
dine conjugated with a thymidine (dT), 2’-deoxycytidine (dC,
detected as the deaminated product where the dC moiety was
converted into a 2’-deoxyuridine, dU), and, to a lower extent,
2’-deoxyadenosine (dA, Figure 3 and see Figures S3–S9 in the
Supporting Information).
Scheme 2. The proposed structures of the ICL products and the
Figure 3. Selected-ion chromatograms (SICs) for monitoring the indi-
cated transitions for the dU-1 cross-link (A) and dT-1 cross-link (B) in
the digestion mixture of ICL products generated from duplex 7
proposed major fragmentation pathways for the [M+H]+ ion of
dU-1 (A) or dT-1 cross-link (B) observed in LC-MS/MS.
*
(7a 6b).
that a [2+2] photocycloaddition reaction occurred, thereby
forming adduct 16 or 17 (Scheme 2). The MS/MS analysis for
monitoring the [M + H]+ ion of dU-1 revealed two major
peaks eluting at 25.6 and 26.9 min in the selected-ion
chromatogram (SIC) for the m/z 696 (16) !580 (18)
transition, which monitors the loss of a neutral 2-deoxyribose
(Scheme 2A). Product ions of m/z 456 (19) and 464 (20) were
also found in the MS/MS as a result of the losses of neutral 2-
deoxyribose and thymine or the second 2-deoxyribose (see
Figure S6A,B,E in the Supporting Information). A similar
fragmentation pathway was observed for dT-1 cross-linking
product 17 (Scheme 2B and see Figure S6C,D,F in the
Supporting Information). It is precedented that a [2+2]
cycloaddition reaction occurred between dT and psoralen
involving either the 3,4-double bond of the pyrone ring or the
4’,5’-double bond of the furan ring.[25] However, the structure
of dA-1 adduct could not be determined at this point. In this
vein, it is worth noting that, upon exposure to 254 nm UV
light, thymine was found to undergo [2+2] cycloaddition with
its neighboring 3’-adenine to give intrastrand cross-link
products.[33,34]
When 1 is opposite to four different nucleosides, the ICL
yields decreased in the order dT (10, 69%) > dC (9, 55%) >
dA (7, 43%) > dG (11, 24%; Figure 1). This result indicates
that 1 has a higher reactivity towards pyrimidines than
purines. To fully investigate the reactivity of 1 towards the
four canonical nucleosides, we synthesized DNA duplexes 12–
15 with only dA, dT, dG, or dC surrounding the photo-cross-
linking site and examined the cross-linking efficiency and the
rate constant for ICL formation. Among these duplexes, the
highest ICL yield (87%) was observed with duplex 13 (dT),
while no ICLs were found with duplex 14 (dG) (Figure 1).
Although duplexes 12 (dA) and 15 (dC) resulted in about 4%
and 7% ICLs, respectively, the reaction of dC or dA with 1 is
much slower than that of dT with 1. The rate constants are in
the order kdT (3.5 ꢀ 0.5 ꢀ 10ꢁ3) > kdC (1.2 ꢀ 0.2 ꢀ 10ꢁ3) > kdA
(0.8 ꢀ 0.2 ꢀ 10ꢁ3; see Figure S10A–C in the Supporting Infor-
mation). All these results supported that dT has a much
higher reactivity than dC and dA towards the coumarin
moiety. The LC-MS/MS analysis of the ICL products obtained
from duplex 7 or 9 showed that the coumarin-derived
thymidine mainly conjugated with dT in duplex 7 and with
dC in duplex 9 (Figure 3 and see Figures S4 and 5 in the
Supporting Information). In addition, all cross-linked prod-
ucts are stable to heating in phosphate buffer or 1.0m
piperidine (see Figure S11 in the Supporting Information).
Collectively, based on the LC-MS/MS analysis and greatly
decreased fluorescence intensity of the ICL adducts and the
relative reactivities of 1 towards dT, dC, and dA, we propose
The ICL formation induced by 1 is photoswitchable.
Irradiation of duplex 10 at 350 nm efficiently generated ICLs,
while the cross-linked product can be split into single-
stranded DNAs by irradiation at 254 nm (Figure 4 and see
Figure S12 in the Supporting Information). Cleavage of the
ICL products to two single-stranded ODNs was ultrafast and
complete within 60 s by irradiation at 254 nm (kcleave = 8.0 ꢀ
0.5 ꢀ 10ꢁ2 sꢁ1, t1/2 = 9.0 s; see Figure S14A in the Supporting
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The signals for the dT-1 and dU-1 cross-links were decreased
from ICL-1 to ICL-2 and ICL-20, whereas a significant
elevation of the dA-1 signal was observed from ICL-1 and
ICL-2 to ICL-20. These results suggested that the formation
of dC-1 and dT-1, but not that of dA-1, is reversible. In this
context, it is worth noting that, as a result of the differences in
the ionization efficiencies for these dinucleosides (dA-1,
because of the higher proton affinity of adenine than uracil or
thymine, is expected to have much better ionization efficiency
than dT-1 and dU-1 in the positive-ion mode), the ratios of the
peak areas displayed in Figure S27 (see the Supporting
Information) do not reflect the relative levels of dT-1, dU-1,
and dA-1 interstrand cross-links. To further substantiate this
finding, we examined the reversibility of ICL formation from
duplexes 12, 13, and 15. Our data showed that the formation
of cross-linked products from duplexes 13 (dT-1 cross-link)
and 15 (dC-1 cross-link) on irradiation at 350 nm were
reversible by irradiation at 254 nm (see Figure S14B,C in the
Supporting Information), while ICL products generated from
duplex 12 (dA-1 cross-link) were not cleaved by irradiation at
254 nm (see Figure S14D in the Supporting Information).
The reversible photo-cross-linking reaction with duplexes 13
and 15 is ultrafast and complete within 90 s and 50 s,
Figure 4. Reversibility of the DNA interstrand photo-cross-linking by
a coumarin-modified ODN duplex 10 over six cycles of 50 min UV
irradiation at 350 nm and 6 min at 254 nm.
Information). This process was highly reversible, and consis-
tent switching behavior was validated over six cycles of
irradiation at 350 nm for 50 min and 254 nm for 6 min
(Figure 4 and see Figure S12 in the Supporting Information).
A similar phenomenon was observed with duplex 7. This
process was also confirmed by mass spectrometry. The
MALDI-TOF-MS analysis showed that the cross-linked
DNA products isolated from the duplex 7 irradiated at
350 nm were split into two single-standed ODNs upon
irradiation at 254 nm with UV light. The detected masses of
two ODNs are 5185 and 4800 Da, which are identical to the
coumarin-modified ODN 7a (calcd: 5186.4 Da) and the
complementary strand 6b (calcd: 4800.2 Da; see Figure S13
in the Supporting Information). So far, neither unsubstituted
nor substituted pyrimidine nucleosides have ever been
applied as components of DNA photoswitches.[35,36] This
feature would be one of the important milestones for
photoswitchable interstrand cross-linking of DNA. The
photochromic behavior of these compounds was observed
by gel electrophoresis analysis followed by UV photoirradia-
tion. Six distinct absorption bands were investigated to verify
the ratification of the exceptional photoswitchability.
To determine whether dT-1, dC-1, and dA-1 cross-links
are reversible, we isolated the ICLs formed from duplex 10
after one cycle of irradiation at 350 nm (ICL-1), one cycle of
irradiation at 350 nm/254 nm (ICL-2), and those formed after
10 cycles of irradiation at 350 nm/254 nm (ICL-20). We then
digested the isolated ICL products with enzymes to release
the ICL as a dinucleoside remnant and subjected the resulting
mixture to LC-MS and MS/MS analyses, as described above.
We then plotted the ratios of the peak areas found in the SICs
to monitor the loss of a 2-deoxyribose from the [M + H]+ ions
of dC-1 (as its deaminated form, that is, 16, m/z 696!580
transition), dT-1 (17, m/z 710!594 transition), and dA-
1 adducts (m/z 710!594 transition) versus that of the [M +
H]+ ion of dA (see Figure S27 in the Supporting Information).
respectively (duplex 13: kdT-1-cleave = 5.0 ꢀ 0.9 ꢀ 10ꢁ2 sꢁ1, t1/2
=
13.0 s; duplex 15: kdC-1-cleave = 8.8 ꢀ 0.4 ꢀ 10ꢁ2 sꢁ1, t1/2 = 8.0 s;
see Figure S14B,C in the Supporting Information). These
data are consistent with our results from LC-MS and MS/MS
analysis, and are also in line with the fact that the cyclobutane-
type photoproduct formed between thymine and the psoralen
moiety can be photoreversed upon irradiation at 254 nm,[37]
whereas the UVC-induced dimeric TA photoproduct cannot
be photoreversed.[34]
In conclusion, we have developed a novel strategy for
photoswitchable interstrand cross-linking of DNA using
a coumarin-modified nucleoside (1) that can be easily
prepared through “click” chemistry. Irradiation at 350 nm of
ODNs containing 1 produced a nonfluorescent DNA cross-
linking product which can be reverted to the original
fluorescent ODNs by irradiation at 254 nm. The mechanism
involves the [2+2] photocycloaddition between coumarin
moiety and dT, dC, or to a much lower extent dA. The dT-
1 and dC-1 adducts are photoreversible, while the dA-
1 adduct is not reversible. The reversible interstrand photo-
cross-linking reaction is bioorthogonal, does not require
additional chemical reagents, and can be controlled in space
and time by choosing different irradiation wavelengths. We
envision that the method is applicable for in situ DNA
manipulation, such as photocontrolled inhibition of DNA
transcription or DNA ligation and the regulation of DNA
aptamers.[38] It provides a simple and versatile approach for
developing reversible DNA fluorescent photoswitches, pho-
toswitchable DNA nanomotors, and smart DNA nanostruc-
tures and nanodevices.
Received: December 6, 2013
Revised: March 19, 2014
Published online: && &&, &&&&
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Communications
DNA Cross-Linking
M. M. Haque, H. Sun, S. Liu, Y. Wang,
X. Peng*
&&&& — &&&&
Photoswitchable Formation of a DNA
Interstrand Cross-Link by a Coumarin-
Modified Nucleotide
A [2+2] cycloaddition reaction between
a coumarin-modified pyrimidine nucleo-
side and the opposing thymidine, 2’-
deoxycytidine, or 2’-deoxyadenosine on
a DNA strand generates photoswitchable
interstrand cross-links (ICLs) in the DNA.
6
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