Synthesis and Photophysical Characterisation of a Fluorescent Nucleoside Analogue that Signals the Presence of an Abasic Site in RNA

Article abstract of DOI:10.1002/cbic.201200408

The synthesis and site-specific incorporation of an environment-sensitive fluorescent nucleoside analogue (2), based on a 5-(benzofuran-2-yl)pyrimidine core, into DNA oligonucleotides (ONs), and its photophysical properties within these ONs are described. Interestingly and unlike 2-aminopurine (a widely used nucleoside analogue probe), when incorporated into an ON and hybridised with a complementary ON, the emissive nucleoside 2 displays significantly higher emission intensity than the free nucleoside. Furthermore, photophysical characterisation shows that the fluorescence properties of the nucleoside analogue within ONs are significantly influenced by flanking bases, especially by guanosine. By utilising the responsiveness of the nucleoside to changes in base environment, a DNA ON reporter labelled with the emissive nucleoside 2 was constructed; this signalled the presence of an abasic site in a model depurinated sarcin/ricin RNA motif of a eukaryotic 28S rRNA. Environmentally sensitive: An environment-sensitive fluorescent nucleoside analogue based on a 5-(benzofuran-2-yl)pyrimidine core was synthesised. When incorporated into a DNA oligonucleotide reporter, it signals the presence of an abasic site in a model depurinated sarcin/ricin RNA motif of eukaryotic 28S rRNA (see figure).

Full text of DOI:10.1002/cbic.201200408

DOI: 10.1002/cbic.201200408
Synthesis and Photophysical Characterisation of
a Fluorescent Nucleoside Analogue that Signals the
Presence of an Abasic Site in RNA
Arun A. Tanpure and Seergazhi G. Srivatsan*[a]
The synthesis and site-specific incorporation of an environ-
ment-sensitive fluorescent nucleoside analogue (2), based on
a 5-(benzofuran-2-yl)pyrimidine core, into DNA oligonucleo-
tides (ONs), and its photophysical properties within these ONs
are described. Interestingly and unlike 2-aminopurine (a widely
used nucleoside analogue probe), when incorporated into an
ON and hybridised with a complementary ON, the emissive nu-
cleoside 2 displays significantly higher emission intensity than
the free nucleoside. Furthermore, photophysical characterisa-
tion shows that the fluorescence properties of the nucleoside
analogue within ONs are significantly influenced by flanking
bases, especially by guanosine. By utilising the responsiveness
of the nucleoside to changes in base environment, a DNA ON
reporter labelled with the emissive nucleoside 2 was construct-
ed; this signalled the presence of an abasic site in a model
depurinated sarcin/ricin RNA motif of a eukaryotic 28S rRNA.
Introduction
Fluorescent nucleoside analogue probes are very useful bio-
physical tools for investigating the structure and binding prop-
erties of nucleic acids.[1] Unlike many proteins that show intrin-
sic fluorescence due to the presence of environment-sensitive
fluorescent aromatic amino acids (e.g., tryptophan), nucleic
acids lack intrinsic fluorescence, as natural nucleobases are
essentially nonemissive.[2] Consequently, several nucleoside an-
alogue probes with useful fluorescence properties have been
developed by 1) using naturally occurring fluorescent heterocy-
cles and polycyclic aromatic hydrocarbons (PAHs) as nucleo-
base surrogates, 2) attaching known fluorophores to the nucle-
obases or sugars (e.g., pyrene, anthracene, phenanthroline, bi-
pyridine and terpyridine), and 3) extending the p conjugation
by attaching heterocycles to nucleobases.[1e,3] For example,
pterdines, a class of naturally occurring planar heterobicyclic
compounds that are structurally similar to purines, have been
used as nucleobase surrogates to develop highly emissive nu-
cleoside analogues.[4] In a similar approach, Kool and co-work-
ers developed PAH base analogues in which the nucleobases
were replaced with fluorescent PAHs (e.g. pyrene, perylene and
phenanthrene).[1c] Such highly nonpolar analogues lacking the
Watson–Crick hydrogen-bonding face have been effectively
implemented in investigating the shape-complementarity re-
quirement in base-pair formation, the electron transfer process
in nucleic acids, and to promote intercalation.[5–7] Fusing addi-
tional aromatic rings onto pyrimidines and purines also enhan-
ces p conjugation, thus generating “size-expanded” fluorescent
nucleobase analogues.[1f] The Saito group has developed sever-
al such fluorescent ring-expanded nucleobases for homogene-
ous single nucleotide polymorphism (SNP) analysis.[1a]
they minimally perturb the structure and function of target oli-
gonucleotides (ONs).[1e] In particular, 2-aminopurine (2-AP), an
isomorphic adenosine analogue that is highly responsive to
changes in its local environment, is one of the most extensive-
ly used nucleoside analogues in nucleic acid-based diagnostics
and discovery assays.[8,9] Pyrrolo-C (pC) and more recently, its
derivative 6-phenylpyrrolo-C (PhpC) have been introduced as
isosteric cytosine (C) analogues.[10,11] As free nucleosides they
display moderate quantum yields, but these drop drastically
when the nucleosides are placed in a double-stranded oligonu-
cleotide. This property of pC and PhpC has been elegantly
used in monitoring RNA secondary structure, and activity of
the RNase H enzyme, respectively.[10,11] In another design strat-
egy, structurally non-invasive pyrimidine analogues have been
developed by attaching or fusing five- or six-membered heter-
ocyclic rings onto pyrimidines. Some of these analogues dis-
play favourable photophysical properties and have been effec-
tively implemented in assays to probe nucleic acid conforma-
tion and function.[12]
Although a significant number of environment-sensitive
base-modified nucleoside analogues with improved fluores-
cence properties have been developed,[13,14] only a very few
nucleosides that retain reasonable quantum yield when incor-
porated into ONs have been implemented in biophysical
assays to study the dynamics and functions of nucleic acids.[15]
Therefore, we sought to develop environment-sensitive nu-
[a] A. A. Tanpure, Dr. S. G. Srivatsan
Department of Chemistry
Indian Institute of Science Education and Research
900, NCL Innovation Park, Dr. Homi Bhabha Road
Pune 411008 (India)
While the majority of the above analogues structurally devi-
ate from the natural nucleosides, isomorphic analogues have
a similar size, shape and Watson–Crick hydrogen bonding face
as that of the native bases, and hence, when incorporated,
E-mail: srivatsan@iiserpune.ac.in
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200408.
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Fluorescent Nucleoside Analogue Probe
cleoside analogue probes that exhibit minimal structural per-
turbation and display emission in the visible region with an
appreciable quantum yield when incorporated into ONs.
By drawing inspiration from a naturally occurring fluorescent
amino acid (tryptophan), we have developed a focused series
of base-modified emissive pyrimidine ribonucleoside ana-
logues by tagging indole, N-methylindole, benzothiophene or
benzofuran at the 5-position of uracil (Figure S1).[16] In particu-
lar, benzothiophene- and benzofuran-conjugated uridine deriv-
atives are reasonably emissive, with emission maxima in the
visible region, and display excellent fluorescence solvato-
chromism. We hypothesised that the responsiveness of these
ribonucleoside analogues to changes in polarity and the neigh-
bouring base environment (when incorporated into RNA ONs)
could also be employed to explore the structure and recogni-
tion properties of DNA ONs. In order to investigate the photo-
physical consequences of placing the emissive nucleoside
within a DNA ON, we chose to incorporate the 2’-deoxy ver-
sion of the benzofuran-conjugated pyrimidine analogue.[17]
Here, we describe the synthesis, photophysical characterisation
and incorporation of benzofuran-conjugated 2’-deoxyuridine
analogue 2 into DNA ONs. Remarkably, upon incorporation
Scheme 1. Sequence of the sarcin/ricin hairpin motif of ribosomal RNA. The
conserved residues are shown in bold. RIP toxins catalytically depurinate res-
idue A4324 to produce the depurinated RNA motif. The structure of the RNA
abasic site (X) is also shown.
the formation of abasic sites in RNA.[24] Nevertheless, abasic
site detection assays that are compatible with screening for-
mats are highly desirable for the discovery of RIP inhibitors.
into single-stranded and double-stranded oligonucleotides the Results and Discussion
emissive nucleoside shows significantly enhanced emission in-
Synthesis and photophysical characterisetion of nucleoside
tensity compared with the free nucleoside, a property that is
rarely displayed by most of the fluorescent nucleoside ana-
logues. Furthermore, by using fluorescence spectroscopy we
investigated the photophysical behaviour of the emissive nu-
cleoside incorporated into DNA ONs in different base environ-
ments. Finally, as proof of the responsiveness of the nucleoside
to environmental changes, we describe the ability of a DNA
ON reporter labelled with modified nucleoside 2 to signal the
presence of an abasic site in a model depurinated sarcin/ricin
RNA motif of a eukaryotic 28S rRNA.
2
The fluorescent deoxyuridine analogue, 5-(benzofuran-2-yl)-2’-
deoxyuridine 2 was synthesised under typical Stille cross-cou-
pling reaction conditions by reacting 5-iodo-2’-deoxyuridine
1 with 2-(tri-n-butylstannyl)benzofuran in the presence of a pal-
ladium catalyst, Pd(PPh3)2Cl2 (Scheme 2).[25] Many fluorescent
nucleoside analogue probes that have been utilised for study-
ing the structure of nucleic acids photophysically respond to
solvent polarity and viscosity changes.[1e] Therefore, before in-
corporation into oligonucleotides, the photophysical properties
of nucleoside 2 were evaluated by performing UV absorption,
and steady-state and time-resolved fluorescence spectroscopic
measurements in solvents of differing polarity and viscosity.
The ground-state electronic spectrum of 2 in water revealed
distinct absorption bands at 265, 272 and 322 nm (Figure 1).
When measured in different solvents, the absorption maxima
of 2 were marginally affected by solvent polarity. However, the
excited-state electronic properties were substantially influ-
enced by changes in solvent polarity. Upon excitation of the
nucleoside at its lowest energy maximum (322 nm) in aqueous
solution, it displayed a strong emission band in the visible
region (lem =446 nm, Figure 1, Table 1). As the solvent polarity
was gradually decreased (water to dioxane), the nucleoside ex-
hibited up to nearly 2.5-fold quenching in fluorescence intensi-
ty and a significant hypsochromic shift (from 446 to 406 nm).
Furthermore, the quantum yields determined in various sol-
vents followed a similar decreasing trend as that for fluores-
cence intensity (Table 1). A positive correlation between the
Stokes shift determined in various solvents and Reichardt’s mi-
croscopic solvent polarity parameter, ET(30), also revealed the
sensitivity of the nucleoside to changes in solvent-polarity en-
vironment (Figure 2).[27]
Unlike DNA abasic sites, which are common DNA lesions,
RNA abasic sites are rare; they are almost uniquely associated
with the depurination activity of ribosome inactivating protein
(RIP) toxins.[18] These toxins (e.g., ricin and saporin) arrest the
function by binding to a highly conserved RNA motif in eu-
karyotic 28S rRNA (the “sarcin/ricin loop”) that interacts with
elongation factors essential for protein synthesis, and depuri-
nating a specific adenosine residue (A4324) in the loop
(Scheme 1).[19] Consequently, the elongation factors necessary
for translation no longer bind to the depurinated ribosome,
thereby leading to cell death.[20] In particular, ricin has been
considered as a potential bioterrorism agent because of its
high toxicity, easy procedures for isolation from natural sources
and the lack of effective treatment against its exposure.[21] Tra-
ditionally, RIP toxins have been detected by using enzyme-
linked immunosorbent assays (ELISAs) and antibody-based im-
munoassays.[22] Methods have also been developed to monitor
the specific depurination activity of RIP toxins by radiolabel-
ling, immunoaffinity chromatography, electro-chemilumines-
cence and mass spectrometry.[23] Usually these methods are la-
borious and involve elaborate assay setups and radiolabelling
procedures. Alternatively, a few abasic-site-sensitive fluores-
cence probes have provided effective tools to directly detect
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S. G. Srivatsan and A. A. Tanpure
Figure 2. Plot of Stokes shift versus ET(30), a microscopic solvent polarity
parameter.
The effect of solvent polarity on the excited-state decay ki-
netics of nucleoside 2 was also investigated by time-resolved
fluorescence spectroscopy. The nucleoside had the highest ex-
cited-state lifetime in water (2.38 ns), and this decreased signif-
icantly in dioxane (0.33 ns, Table 1, Figure S2). The ratio of radi-
ative (kr) to nonradiative (knr) decay rate constants, determined
by lifetime and quantum yield in different solvents, revealed
that the radiative pathway is considerably favoured in polar
solvents compared with nonpolar solvents (Table 1).
Scheme 2. Synthesis of fluorescent nucleoside, 1-(2-deoxy-b-d-ribofurano-
syl)-5-(benzofuran-2-yl)uracil 2 and the corresponding phosphoramidite sub-
strate 4.[25] a) Pd(PPh3)2Cl2, dioxane, 908C, 97%; b) 4,4’-dimethoxytritylchlor-
ide (DMT-Cl), 4-dimethylaminopyridine (DMAP), pyridine, RT, 67%;
c) iPr2NP(Cl)OEtCN, iPr2NEt, CH2Cl2, RT, 51%.
The relative conformation of the pyrimidine ring and conju-
gated aromatic ring separated by a rotatable aryl–aryl bond in
modified nucleoside analogues, which has a direct impact on
the conjugation and, hence, fluorescence properties, has been
shown to be sensitive to molecular crowding and viscosity.[28]
In comparison with low-viscosity media, viscous media restrict
free rotation, thereby resulting in structural rigidification and
usually enhanced fluorescence emission.[29] When incorporated
into ONs, a conjugated nucleoside analogue can undergo
structural rigidification–derigidification by interactions with
neighbouring bases during a folding or recognition process.[28]
In order to assess the effects of changes in conformation of
the benzofuran moiety relative to the nucleobase, further fluo-
rescence characterisation of nucleoside 2 were performed in
solvents of similar polarity but differing viscosity. As the sol-
vent viscosity increased, from ethylene glycol (h258C =16.1 cP)
to glycerol (h258C =934 cP), the nucleoside shows enhancing
emission intensity (nearly 1.5-
Figure 1. Absorption (c) and emission (a) spectra of nucleoside 2 (25
and 5.0 mm, respectively) in different solvents. FI=fluorescence intensity. For
fluorescence studies, samples were excited at 322 nm, and excitation and
emission slit widths were 3 and 5 nm, respectively.[26]
fold higher in glycerol, relative
to ethylene glycol) with no ap-
Table 1. Photophysical properties of 2 in different solvents.[25,26]
parent change in emission maxi-
[b]
Solvent
lmax[a] [nm]
lem [nm]
Irel
F[c]
tave[c] [ns]
kr/knr
mum (Figure 3 and Table S1).
Lifetime and anisotropy meas-
urements also revealed a longer
decay time and a higher aniso-
tropy value in glycerol than in
ethylene glycol, which is consis-
tent with the viscosity difference
water
322
322
322
322
446
423
411
406
1.0
0.8
0.3
0.4
0.19
0.12
0.04
0.07
2.38
0.78
0.27
0.33
0.24
0.13
0.04
0.08
methanol
acetonitrile
dioxane
[a] lmax corresponding to the lowest energy maximum is given. [b] Relative emission intensity is given relative
to the intensity in water. [c] Standard deviations for F and tave were ꢀ0.002 and 0.02 ns, respectively.
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Fluorescent Nucleoside Analogue Probe
confirmed by mass analysis (Figure S3, Table S2). The presence
of a benzofuran moiety can potentially perturb the structure of
an ON, and hence the formation of a stable duplex with its
complementary ON. The stability of a duplex assembled by hy-
bridising one of the modified ONs (8) with its complementary
ON (8c) was studied by a UV-thermal denaturation experiment.
This showed only a small difference in Tm between the modi-
fied duplex 8·8c and the unmodified duplex 9·8c, thus indicat-
ing that the benzofuran modification has an only marginal
effect on duplex stability (Figure S4, Table S3).
Photophysical characterisation of nucleoside 2 in different
base environment
The photophysical properties of fluorescent nucleosides incor-
porated into ONs can be altered by a variety of mechanisms,
such as stacking of the chromophore to flanking bases, colli-
sional and hydrogen-bond interactions with neighbouring
bases, the solvation–desolvation effect, rigidification–derigidifi-
cation of the chromophore and excited-state processes with
neighbouring bases.[8,28,30,31] The influence of neighbouring
bases on the fluorescence properties of 2 was studied by per-
forming steady-state fluorescence measurements with DNA
ONs 5–8 and duplexes constructed by hybridising 5–8 with
their respective complementary ONs (5c–8c, Figure 4). Single-
stranded ONs 5–7 (emissive nucleoside between dA, dT or dC
residues, respectively) exhibited significantly enhanced emis-
sion compared with the free nucleoside (Figure 5). Interesting-
Figure 3. Emission spectra of nucleoside 2 (5.0 mm) in solvents of varying vis-
cosity. FI=fluorescence intensity. Samples were excited at 322 nm, and exci-
tation and emission slit widths were 1 and 10 nm, respectively.[26]
between the solvents (Table S1). Taken together, emission in
the visible region with a reasonable quantum yield and the
dual-sensitivity of the nucleoside fluorescence to solvent polar-
ity and viscosity prompted us to study the responsiveness of
the emissive nucleoside within DNA ONs.
Incorporation of nucleoside 2 into DNA ONs
The phosphoramidite substrate 4, which is necessary for the
solid-phase ON synthesis, was prepared by first protecting the
5’-hydroxyl with a dimethoxytrityl group, followed by phosphi-
tylation of the 3’-hydroxyl in the presence of 2-cyanoethyl di-
isopropylchlorophosphoramidite (Scheme 2).[25] To study the
effect of flanking bases on the fluorescence of the modified
nucleoside, a series of DNA ONs (5–8) was synthesised in
which 2 was placed between different bases (Figure 4). The
modified phosphoramidite was incorporated into 17-mer DNA
ONs under standard solid-phase ON synthesis conditions. The
deprotected ONs were then purified by PAGE under denatur-
ing conditions. The integrity of full-length modified ONs was
Figure 5. Fluorescence intensity (FI) of modified ONs (5–8, 1 mm) and cor-
responding duplexes (1 mm) in 20 mm cacodylate buffer (pH 7.0, 100 mm
NaCl, 0.5 mm EDTA) at their respective emission maxima (422–444 nm, see
Table S4). Samples were excited at 330 nm; excitation and emission slit
widths were 3 and 10 nm, respectively.[25,26]
ly, when incorporated into duplexes 5·5c and 6·6c, nucleoside
2 showed enhanced emission compared with the respective
single-stranded ONs (Figure 5, Figure S5). This observation is
particularly noteworthy because the majority of emissive nu-
cleoside analogues (e.g., 2-AP, pyrroloC)[10,32] show progressive
fluorescence quenching upon incorporation into single-strand-
ed and double-stranded ONs, a major impediment to the use
of many such analogues in in vivo assays.
Figure 4. Sequences of fluorescently modified (5–8) and custom synthesised
(9–12 and 5c–8c) DNA oligonucleotides (9 is a control: unmodified DNA).
While hybridisation of 5–8 with 5c–8c, respectively, places nucleoside 2 op-
posite its complementary base, hybridisation of 8 with 10–12 places 2 op-
posite mismatched bases.[26]
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S. G. Srivatsan and A. A. Tanpure
However, ON 8, in which 2 is flanked by guanosine residues,
displayed a slightly blue-shifted and markedly less-intense (ca.
threefold) emission band compared with the free nucleoside 2
(Figures 5 and S5). The quenching effect was more pro-
nounced (~tenfold) when the emissive nucleoside was placed
opposite the complementary base in the “perfect” duplex
8·8c. 2-AP and several other fluorescent nucleoside analogues
exhibit similar fluorescence intensity quenching when incorpo-
rated into ONs.[1e,8,32] Furthermore, duplexes 8·10, 8·11 and
8·12, in which the emissive nucleoside is opposite mismatched
bases, showed similar fluorescence quenching to that of the
“perfect” duplex 8·8c (Figure 6). This fluorescence quenching,
a pyrene-modified deoxyribonucleotide triphosphate, which
was preferentially incorporated opposite an abasic site by DNA
polymerase, to identify the presence of abasic sites.[5,39] Alter-
natively, fluorescence-based methods with probes that show
changes in their emission properties when adjacent or oppo-
site abasic sites were found to be more useful as they offered
direct detection of abasic sites in DNA and RNA.[24,40,41] Shipova
and Gates developed one of the first fluorimetric assays to
monitor the time-depended generation of abasic sites by
using 2-AP-labelled DNA hairpin constructs.[41] In this study 2-
AP was placed adjacent to a guanine residue that was alkylat-
ed by leinamycin[42] and subsequently excised to produce an
abasic site adjacent to the fluorescent probe. Upon treatment
with leinamycin, 2-AP positively reported the formation of
abasic sites with a significant enhancement in fluorescence
intensity. By adopting a similar approach, pyrene- and thio-
phene-modified fluorescent nucleoside analogues and a fluo-
rescent ligand that specifically binds to an abasic site were
used in monitoring the formation of abasic sites in RNA oligo-
nucleotides.[24]
Our previous study revealed that a short RNA ON reporter
containing the benzofuran-conjugated ribonucleoside ana-
logue could specifically signal the presence of a DNA abasic
site in an RNA–DNA heteroduplex.[16a] This observation encour-
aged us to study the impact of placing the deoxyribonucleo-
side 2 opposite an RNA abasic site. As before, a series of DNA–
RNA heteroduplexes was assembled by annealing modified
DNA ONs 5–8 to the respective complementary RNA ONs 13c–
16c or to ONs 13a–16a containing a chemically stable abasic
site surrogate, tetrahydrofuran (Figures 4 and 7A). Nucleoside
2 (flanked by dA, dT or dC), when placed opposite an abasic
site in duplexes 5·13a, 6·14a or 7·15a, showed discernible
quenching in fluorescence intensity compared with the corre-
sponding “perfect” duplexes 5·13c, 6·14c and 7·15c (Fig-
ure 7B). However, duplex 8·16c and abasic-site-containing
duplex 8·16a, in which the nucleoside is flanked by dG resi-
dues, exhibited very weak fluorescence.[33]
Figure 6. Emission spectra of 2, 8 and duplexes assembled by hybridising 8
with complementary (8c) and mismatched ONs (10–12). FI=fluorescence in-
tensity. Samples were excited at 330 nm; excitation and emission slit widths
were 7 and 10 nm, respectively.[26]
along with a small spectral blue shift, might be attributable to
a combination of the following: an electron transfer process
between the fluorescent nucleoside and adjacent guanosine
residues,[30,31,33] a desolvation effect[8] and alterations in the
conformation of the benzofuran moiety relative to the nucleo-
base.[28] These results clearly reveal the influence of neighbour-
ing bases on the fluorescence of nucleoside 2. Such a property
has been employed in devising nucleic acid-based molecular
beacons and in the detection of a specific mismatch in DNA
duplexes.[1a,34]
To further assess the ability of emissive nucleoside to detect
the presence of an abasic site in a biologically relevant RNA
motif, we decided to use model RNA ONs 17 and 18 (Fig-
ure 8A). ON 17, which contains the conserved sarcin/ricin loop
region of eukaryotic 28S rRNA, is a commonly used substrate
to study the depurination activity of RIPs.[23b,24] ON 18 contains
a chemically stable abasic site substitute, tetrahydrofuran, and
acts as a model of the depurinated product that would be ob-
tained upon depurination of substrate 17 by RIP toxins.[24b,d]
A
complementary DNA ON 19 labelled with 2 was synthesised;
upon hybridisation to 17 or 18, this would place 2 opposite
a complementary base and an abasic site, respectively (Fig-
ure 8A).[43] A duplex of fluorescent ON and RIP RNA substrate
(19·17) showed a strong emission band, the intensity of which
was nearly 1.5-fold higher than that for single-stranded 19
(Figure 8B). Interestingly, the duplex of ON probe and depuri-
nated product mimic (19·18) exhibited drastically quenched
emission (ca. eightfold) relative to that of duplex 19·17 (Fig-
ure 8B). Although the exact morphology of the nucleoside 2 in
19·18 is unknown, we believe that the observed quenching in
Fluorescence detection of an RNA abasic site
Early methods to detect abasic sites were largely focused on
the detection of abasic sites in DNA as these are formed spon-
taneously or as intermediates in the base-excision repair pro-
cess of damaged nucleobases.[35,36] These methods relied on
the specific and irreversible reaction between aldehyde-reac-
tive probes and abasic sites of isolated DNA.[37] Later, Green-
berg developed a more sensitive method by utilising biotiny-
lated cysteine to specifically detect the oxidised form of an
abasic lesion, 2-deoxyribonolactone.[38] Matray and Kool used
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Fluorescent Nucleoside Analogue Probe
Figure 7. A) Sequences of synthetic RNA oligonucleotides. While hybridisa-
tion of 5–8 with 13c–16c, respectively, places nucleoside 2 opposite its
complementary base, hybridisation of 5–8 with 13a–16a places it opposite
a chemically stable abasic site surrogate X. B) Fluorescence intensity (FI) of
heteroduplexes (1 mm) in 20 mm cacodylate buffer (pH 7.0, 100 mm NaCl,
0.5 mm EDTA) at 430 nm. Samples were excited at 330 nm; excitation and
emission slit widths were 3 and 10 nm, respectively.[25,26]
Figure 8. A) Sequence of synthetic RNA (17, RIP substrate) and depurinated
product mimic (18) containing a chemically stable abasic site substitute (X).
Adenosine residue A15 (A4324 in rat 28S rRNA) is specifically depurinated by
RIP toxins to produce an abasic site. cDNA (19) containing fluorescent nu-
cleoside 2 is also shown. B) Emission spectra (1 mm) of substrate (19·17) and
depurinated product duplexes (19·18). FI=fluorescence intensity. Samples
were excited at 330 nm; excitation and emission slit widths were 7 and
10 nm, respectively.[25,26]
fluorescence intensity might be attributable to the following
reasons. In duplex 19·17 the benzofuran moiety (tagged at the
5-position of the base) is extrahelical and projects towards the
major groove, and hence it is less stacked and located away
from the guanosine residues of ON 17 (Figure S6). However, in
duplex 19·18 the benzofuran ring is presumably intrahelical,
because 2 opposite an abasic site can potentially undergo
anti-to-syn conformational change as it is not restricted by
complementary base pairing.[28] In this situation the fluoro-
phore is better stacked and closer to the guanosine residues of
18, thereby resulting in quenching of fluorescence.[44] Together,
it can be inferred that 2 in duplex 19·18 signals the presence
of an abasic site in RNA, albeit with quenched emission. This
attribute of the emissive nucleoside can be potentially imple-
mented in a fluorescence hybridisation assay to detect the
depurination activity of highly toxic RIPs.[24]
the fluorescent analogue incorporated into a DNA ON reporter
to detect the presence of an abasic site in RNA highlights the
potential of 2 as a fluorescent probe. As the conformation of
the emissive nucleoside, its surrounding environment and/or
its interaction with neighbouring bases are likely to be altered
during a folding or recognition event, it is expected that this
environment-sensitive nucleoside analogue will be a useful
probe in studying the structure and function of nucleic acids.
Experimental Section
Photophysical characterisation of benzofuran-conjugated 2’-de-
oxyuridine 2
Conclusions
Steady-state fluorescence in various solvents: Modified nucleoside 2
(5 mm) in water, methanol, acetonitrile or dioxane was excited at its
lowest energy absorption maximum (322 nm), with excitation and
emission slit widths of 3 nm and 5 nm, respectively. In the case of
ethylene glycol and glycerol the excitation and emission slit widths
were 1 nm and 10 nm, respectively. All solutions contained DMSO
(0.5%). Fluorescence experiments were performed in triplicate in
a micro fluorescence cell (path length 1.0 cm; Hellma Optics, Jena,
A microenvironment-sensitive fluorescent 2’-deoxyuridine ana-
logue that is based on a 5-(benzofuran-2-yl)pyrimidine core
and that displays emission in the visible region has been incor-
porated into DNA ONs. Notably, upon incorporation into du-
plexes, nucleoside 2 remarkably maintains its fluorescence effi-
ciency, a property that is seldom exhibited by the majority of
fluorescent nucleoside analogues. Furthermore, the ability of
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S. G. Srivatsan and A. A. Tanpure
Germany) on
(Horiba, Kyoto, Japan).
a FluoroLog-3 fluorescence spectrophotometer
Keywords: abasic site
fluorescent nucleoside · RNA
· DNA · emission spectroscopy ·
Time-resolved fluorescence measurements: Excited-state lifetimes of
2 in various solvents were determined by using a time correlated
single photon counting (TCSPC) fluorescence spectrophotometer
(Horiba). In water, methanol, ethylene glycol and glycerol the con-
centration of 2 was 5 mm; in acetonitrile and dioxane it was
250 mm. In water, methanol, ethylene glycol and glycerol, 2 was
excited by using a NanoLED 339 nm source (IBH/Horiba, Glasgow,
UK) with a band pass of 4 nm, and fluorescence emission was col-
lected. Similarly, 2 in acetonitrile and dioxane was excited by using
a NanoLED 375 nm diode laser source (IBH) with a band pass of
12 nm. Lifetime measurements were performed in duplicate, and
decay profiles were analysed with DAS6 analysis software (IBH).
Fluorescence intensity decay kinetics in water and ethylene glycol
were found to be monoexponential, whereas in other solvents
they were found to be biexponential, with c2 (goodness of fit)
values very close to unity.
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Acknowledgements
S.G.S and A.A.T thank the Department of Science and Technology,
India (SR/S1/OC-51/2009) and the Council of Scientific and Indus-
trial Research, India for financial support and graduate research
fellowship, respectively.
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Received: June 14, 2012
Published online on October 15, 2012
ChemBioChem 2012, 13, 2392 – 2399
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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