A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: Synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site

Article abstract of DOI:10.1002/chem.201101194

Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events. Uridine blue: T7 RNA polymerase effectively incorporates a polarity-sensitive fluorescent pyrimidine ribonucleotide analogue to produce emissive RNA oligonucleotides. The enzymatically generated fluorescent oligoribonucleotide reporter positively signals the presence of a DNA abasic site (see picture).

Full text of DOI:10.1002/chem.201101194

DOI: 10.1002/chem.201101194
A Microenvironment-Sensitive Fluorescent Pyrimidine Ribonucleoside
Analogue: Synthesis, Enzymatic Incorporation, and Fluorescence Detection
of a DNA Abasic Site
Arun A. Tanpure and Seergazhi G. Srivatsan*[a]
Abstract: Base-modified fluorescent ri-
bonucleoside-analogue probes are val-
uable tools in monitoring RNA struc-
ture and function because they closely
resemble the structure of natural nucle-
the 5-position of uracil. One of the an-
alogues, based on a 5-(benzofuran-2-
yl)pyrimidine core, shows emission in
the visible region with a reasonable
quantum yield and, importantly, dis-
plays excellent solvatochromism. The
corresponding triphosphate substrate is
effectively incorporated into oligoribo-
nucleotides by T7 RNA polymerase to
produce fluorescent oligoribonucleo-
tide constructs. Steady-state and time-
resolved spectroscopic studies with flu-
orescent oligoribonucleotide constructs
demonstrate that the fluorescent ribo-
nucleoside photophysically responds to
subtle changes in its environment
brought about by the interaction of the
chromophore with neighboring bases.
In particular, the emissive ribonucleo-
side, if incorporated into an oligoribo-
nucleotide, positively reports the pres-
ence of a DNA abasic site with an ap-
preciable enhancement in fluorescence
intensity. The straightforward synthesis,
amicability to enzymatic incorporation,
and sensitivity to changes in the micro-
environment highlight the potential of
the benzofuran-conjugated pyrimidine
ribonucleoside as an efficient fluores-
cent probe to investigate nucleic acid
structure, dynamics, and recognition
events.
obases. Especially, 2-aminopurine,
a
highly environment-sensitive adenosine
analogue, is the most extensively uti-
lized fluorescent nucleoside analogue.
However, only a few isosteric pyrimi-
dine ribonucleoside analogues that are
suitable for probing the structure and
recognition properties of RNA mole-
cules are available. Herein, we describe
the synthesis and photophysical charac-
terization of a small series of base-
modified pyrimidine ribonucleoside an-
alogues derived from tagging indole,
N-methylindole, and benzofuran onto
Keywords: abasic sites · DNA · flu-
orescent probes · ribonucleosides ·
RNA
Introduction
heterocycles to the bases.[6] By utilizing these design strat-
egies, several classes of base-modified fluorescent nucleo-
base analogues, size-expanded,[9] extended,[6,10] pteridine,[11]
polycyclic aromatic hydrocarbon,[9] and isomorphic,[6] have
been developed. A significant number of these analogues
that display useful photophysical properties have been im-
plemented in DNA-based and, to a relatively lesser extent,
in RNA-based biophysical assays.[12,13] Notably, the majority
of these analogues, except isomorphic bases, substantially
deviate from the native structure of the nucleobases. Iso-
morphic analogues are advantageous because they maintain
the Watson–Crick (WC) base pairing and minimally perturb
the native structure and function of target oligonucleotides.
In particular, a highly emissive and environment-sensitive
adenosine analogue, 2-aminopurine (2AP), has been exten-
sively utilized in designing several DNA- and RNA-based
biophysical and discovery assays.[7,14,15] However, excitation
and emission maxima in the UV region and the drastically
low quantum yields displayed by 2AP when incorporated
into oligonucleotides greatly limit its application to in vitro
systems only.[15,16]
Fluorescent nucleoside analogues that photophysically
report changes in nucleic acid conformation have become
very important bioanalytical tools in advancing the under-
standing of nucleic acid structure and function.[1–7] Nucleic
acids lack intrinsic fluorescence as natural nucleobases are
practically nonemissive.[8] In order to impart better photo-
physical properties to otherwise nonemissive natural nucleo-
bases, most design principles rely on utilizing naturally oc-
curring fluorescent heterocycles or polycyclic aromatic hy-
drocarbons or on extending the p conjugation by appending
[a] A. A. Tanpure, Dr. S. G. Srivatsan
Department of Chemistry
Indian Institute of Science Education and Research
Sai Trinity Building, Pashan, Pune 411021 (India)
Fax : (+91)20-25899790
E-mail: srivatsan@iiserpune.ac.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101194. It contains details of
the synthesis of the modified ribonucleosides, photophysical charac-
terizations of the ribonucleoside analogues and RNA transcripts,
MALDI-TOF MS analyses of the RNA transcripts, quenching studies,
thermal denaturation studies, gel mobility shift experiments, and
NMR and mass spectra of the compounds.
Numerous base-modified ribonucleoside analogues with
emission in the visible region and a high quantum yield
have been developed, but only a few analogues have been
effectively implemented in exploring the functions of RNA
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FULL PAPER
molecules.[7] In particular, only
a very few structurally nonper-
turbing pyrimidine ribonucleo-
side analogues have shown
promise as useful fluorescent
probes.[17–19] In order to expand
the repertoire of useful fluores-
cent pyrimidine ribonucleoside
analogues, we sought to synthe-
size microenvironment-sensitive
pyrimidine ribonucleoside ana-
logues that are structurally non-
perturbing, display emission in
the visible region, and maintain
a
reasonable quantum yield
upon incorporation into oligori-
bonucleotides.
We have drawn inspiration
from a naturally occurring fluo-
rescent amino acid, tryptophan,
to construct a series of emissive
pyrimidine ribonucleoside ana-
logues. Tryptophan, an indole
derivative, is reasonably emis-
sive, and its emission properties
are highly sensitive to its local
environment.[20] Therefore, we
hypothesize that the attachment
of a bicyclic heterocycle such as
an indole moiety to a nucleo-
base (for example, to the 5-po-
Scheme 1. Synthesis of modified ribonucleoside analogues 3–5 and ribonucleotide analogue 6. See the Support-
ing Information for experimental details. Boc: tert-butoxycarbonyl; DMF: N,N-dimethylformamide.
sition of uracil) may enhance the p conjugation and impart
better photophysical properties to the nucleobase.[21,22]
Herein, we describe the synthesis and photophysical charac-
terization of a focused set of new pyrimidine ribonucleoside
analogues derived by tagging indole, N-methylindole, and
benzofuran at the 5-position of uracil. Notably, the benzo-
furan-conjugated uridine analogue is appreciably emissive,
with an emission maximum in the visible region, and it also
exhibits excellent solvatochromism. We also report the syn-
thesis of the corresponding ribonucleoside triphosphate sub-
strate and its effective incorporation into RNA oligonucleo-
tides by transcription reactions catalyzed by T7 RNA poly-
merase. Furthermore, the emissive uridine analogue, when
incorporated into an oligoribonucleotide, positively signals
the presence of a DNA abasic site.
presence of 3m HCl. The N-methylindole- and benzofuran-
conjugated uridines, 4 and 5, have been synthesized by treat-
ing 5-iodouridine (1) with the corresponding stannylated
heterocycles in the presence of a palladium catalyst.
Photophysical characterization of ribonucleoside analogues:
An important attribute exhibited by many conformation-
sensitive fluorescent nucleoside-analogue probes is that they
photophysically respond to changes in solvent polarity.[6]
Therefore, prior to incorporation into RNA oligonucleo-
tides, the photophysical properties have been examined in
solvents of different polarity to evaluate the responsiveness
of the modified ribonucleosides to polarity changes. An in-
crease in solvent polarity has marginal impact on the
ground-state electronic spectrum of modified ribonucleo-
sides 3–5 (Figure 1 and Table 1).[23] However, the quantum
yield, emission maximum, and fluorescence lifetime are sig-
nificantly affected by the solvent polarity. Unexpectedly, the
indole- and N-methylindole-conjugated uridine analogues (3
and 4, respectively) show very weak fluorescence in nonpo-
lar solvents and practically no fluorescence in water (Figur-
es S1 and S2 and Table S1 in the Supporting Information).
Rewardingly, if excited at its lowest energy maximum
(322 nm), the benzofuran-conjugated uridine 5 in water ex-
hibits a very strong emission band (lem =447 nm) that corre-
Results and Discussion
Synthesis of ribonucleoside analogues: Modified uridine an-
alogues 3–5 have been synthesized according to the proce-
dure illustrated in Scheme 1. In a two-step reaction, indole-
conjugated uridine 3 has been synthesized by performing a
palladium-catalyzed cross-coupling reaction between 5-io-
douridine (1) and a tributylstannyl derivative of N-Boc-pro-
tected indole, followed by removal of the Boc group in the
sponds to
a
quantum yield of 0.212Æ0.002 (Figure 1,
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S. G. Srivatsan and A. A. Tanpure
to dioxane, a huge decrease in the lifetime (approximately
sixfold) is observed in dioxane relative to that in water. The
decreasing trend in the lifetime is consistent with the inten-
sity displayed by the ribonucleoside in various solvents. Ra-
diative (kr) and nonradiative (knr) decay rate constants in
different solvents have been calculated by using experimen-
tally determined lifetimes and quantum yields (Table 1). A
nearly 2.5-fold higher kr/knr ratio in water clearly indicates
that the radiative pathway is appreciably favored in water
relative to that in dioxane. Properties such as emission in
the visible region, a modest quantum yield, and responsive-
ness to polarity changes as illustrated by fluorescence meas-
urements adequately confer probelike attributes to the fluo-
rescent ribonucleoside analogue 5. Therefore, ribonucleo-
side 5 has been selected for incorporation into RNA oligo-
nucleotides by transcription reactions. The corresponding ri-
bonucleoside triphosphate substrate 6, which is necessary
for in vitro transcription reactions, has been synthesized by
employing the method developed by Ludwig.[26] The modi-
fied triphosphate 6 has been synthesized in a one-pot two-
step reaction by treating ribonucleoside 5 with freshly dis-
tilled POCl3, followed by a reaction with bistributylammoni-
um pyrophosphate (Scheme 1).
Figure 1. Absorption (25 mm, solid lines) and emission (5.0 mm, dashed
lines) spectra of ribonucleoside 5 in various solvents. Samples were excit-
ed at 322 nm. Excitation and emission slit widths were maintained at 2
and 4 nm, respectively. All solutions for absorption and emission studies
contained 2.5% and 0.5% dimethylsulfoxide (DMSO), respectively.
Table 1. Photophysical properties of fluorescent ribonucleoside analogue
5 in various solvents.[25]
[a]
[b]
[c]
Solvent
lmax
lem
Irel
F[c]
tav
kr/
[d]
Enzymatic incorporation: Although, solid-phase chemical
synthesis is the method of choice for the synthesis of modi-
fied oligoribonucleotides, enzymatic methods such as tran-
scription and ligation reactions have also been effectively
utilized in labeling RNAs.[27–29] In order to investigate the
ability of T7 RNA polymerase to incorporate the modified
triphosphate 6 into oligoribonucleotides, in vitro transcrip-
tion reactions were conceived, as illustrated in Figure 2. A
series of promoter–template duplexes were assembled by
annealing a DNA promoter strand to DNA templates, D1–
D5 (Figure 2). The templates were designed to contain one
or two deoxyadenosine residues at different positions to
direct single or double incorporations of the modified tri-
phosphate 6. Additionally, a deoxythymidine residue was
placed at the 5’-end of each template strand to direct the ad-
dition of a single adenosine at the 3’-end of each transcript.
Hence, in vitro transcription reactions in the presence of
GTP, CTP, UTP/6, and a-32P ATP, if successful, would result
in the formation of full-length oligoribonucleotide tran-
scripts containing a 32P-labeled adenosine at the 3’-end. Re-
action products could then be resolved by analytical dena-
turing polyacrylamide gel electrophoresis and imaged.
Failed transcription reactions that resulted in transcripts
shorter than the full-length products would not carry the
32P-labeled adenosine and, hence, would remain undetected.
A transcription reaction in the presence of template D1
results in the formation of the 10-mer modified full-length
RNA transcript 8 containing the modified ribonucleoside at
the +7 position (Figure 3, lane 2). Along with the full-
length product, trace amounts of N+1 and N+2 products
are also formed due to nontemplated random incorporation
of ribonucleotides.[30] The triphosphate 6 is incorporated
into the oligoribonucleotide with a moderate efficiency of
[nm]
[nm]
[ns]
knr
water
322
322
322
322
447
423
410
404
1.0
0.8
0.4
0.6
0.212
0.145
0.063
0.099
2.55
0.94
0.33
0.43
0.27
0.16
0.06
0.11
methanol
acetonitrile
dioxane
[a] The lowest energy maximum is given. [b] Emission intensity relative
to intensity in water. [c] Standard deviations for the quantum yield (F)
and average lifetime (tav) are ꢀ0.004 and 0.02 ns, respectively. [d] kr and
knr are radiative and nonradiative decay rate constants, respectively.
Table 1). As the solvent polarity is sequentially decreased
from water to dioxane, the ribonucleoside shows a signifi-
cant hypsochromic shift (447 to 404 nm) and a nearly two-
fold quenching in fluorescence intensity. Furthermore, the
relative quantum yield determined by using 2AP as a stan-
dard in various solvents is in good agreement with the ob-
served fluorescence intensity in the respective solvents
(Table 1). The responsiveness of the ribonucleoside to
changes in the solvent-polarity environment is also con-
firmed by plotting the Stokes shift in various solvents as a
function of Reichardtsꢁ microscopic solvent polarity parame-
ter, ET(30) (Figure S3 in the Supporting Information).[24]
A
positive correlation between the Stokes shift and ET(30) fur-
ther establishes the sensitiveness of the emissive ribonucleo-
side 5 to its surrounding environment.
Time-resolved fluorescence spectroscopic measurements
have also been performed to study the effect of solvent po-
larity on the excited-state decay kinetics of ribonucleoside 5
(Table 1, Figure S4 in the Supporting Information). The
decay profile of the emissive ribonucleoside in solvents of
different polarity reveals distinct decay kinetics. The ribonu-
cleoside in water has the highest lifetime of 2.55 ns. As the
polarity decreases when the solvent is changed from water
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RNA product is not produced
(Figure 3, lane 3). This result
shows that the formation of
full-length transcripts is not due
to adventitious misincorpora-
tion. Interestingly, in a reaction
containing UTP and 6 in equi-
molar concentrations, T7 RNA
polymerase incorporates both
the natural and the modified
UTP into RNA transcripts
(Figure 3, lane 4).
In order to test the ability of
T7 RNA polymerase to incor-
porate the modification near
the promoter region, transcrip-
tion reactions were performed
with templates D2 and D3,
which would direct the incorpo-
ration of 6 at the +3 and +4
positions, respectively.[31] The
incorporation efficiencies were
Figure 2. Top: Incorporation of ribonucleoside triphosphate 6 by transcription reactions in the presence of
DNA templates D1–D5. Bottom: Sequences of the synthetic oligonucleotides (9–15) used in this study. “r”
preceding the parentheses with the sequence indicates an RNA oligonucleotide. See the Experimental Section
for details.
(67Æ3)% relative to the reaction in the presence of natural
UTP. Discernible retardation in the mobility of modified
transcript 8 with respect to unmodified transcript 7 clearly
indicates the inclusion of the higher molecular weight emis-
sive ribonucleoside during the transcription reaction
(Figure 3, compare lanes 1 and 2). When neither UTP nor 6
is included in a control transcription reaction, the full-length
found to be lower for reactions with templates D2 and D3
(Figure 3, lanes 6 and 8). Notably, reactions in the presence
of templates D4 and D5 resulted in double incorporation of
6 in successive as well as separate sites (Figure 3, lanes 10
and 12). Albeit with moderate yields, the reactions with var-
ious templates clearly demonstrate the usefulness of in vitro
transcription reactions in generating singly and doubly
modified fluorescent oligoribonucleotides.
Characterization of fluorescent oligoribonucleotide tran-
scripts: Large-scale transcription reactions were performed
in the presence of non-radiolabeled triphosphates and tem-
plate D1 to isolate transcript 8 for further chemical and
photophysical characterizations (Figure S5 in the Supporting
Information). A typical reaction after gel electrophoretic
purification gave nearly 14 nmol of the modified oligoribo-
nucleotide product. The presence of benzofuran-conjugated
uridine
5 in oligoribonucleotide 8 was confirmed by
MALDI-TOF mass analysis (Figure 4). Furthermore, re-
versed-phase HPLC analysis of ribonucleoside products ob-
tained from an enzymatic digestion reaction of transcript 8
clearly revealed the presence of modified ribonucleoside 5
(Figure 5). The ribonucleoside composition determined from
the area under the individual ribonucleoside peak also
matched well with the sequence of the full-length oligoribo-
nucleotide. Mass analysis of HPLC fractions corresponding
to the retention time of each ribonucleoside unambiguously
established the authenticity of the modified ribonucleoside
present in transcript 8 (Table S2 in the Supporting Informa-
tion).[25] Together, these results clearly reveal the incorpora-
tion of the emissive ribonucleotide into RNA oligonucleo-
tides by RNA polymerase in transcription reactions.
Figure 3. Polyacrylamide gel electrophoresis of transcripts obtained from
in vitro transcription reactions with DNA templates D1–D5 in the pres-
ence of UTP and modified UTP 6 under denaturing conditions. The per-
centage incorporation of 6 is reported with respect to the amount of full-
length product formed in the presence of UTP. All reactions were per-
formed in duplicate and the errors in yields were ꢀ4%. See the Experi-
mental Section for details.
Photophysical characterization of fluorescent oligoribo-
AHCTUNGTREGnNNUN ucleotides: The fluorescence properties of emissive nucleo-
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S. G. Srivatsan and A. A. Tanpure
time-resolved fluorescence spectroscopic measurements
have been carried out with oligoribonucleotide 8 and du-
plexes of 8 to evaluate the influence of neighboring bases
on the fluorescence of ribonucleoside 5. A series of duplexes
have been assembled by hybridizing oligoribonucleotide 8
to complementary DNA and RNA oligonucleotides, in
which the ribonucleoside 5 has been placed opposite com-
plementary or mismatched bases (Figure 2).
The photophysical properties of oligoribonucleotide 8
reveal characteristic features that are different from those of
the free ribonucleoside 5 in aqueous buffer. Upon excita-
tion, the single-stranded oligoribonucleotide 8 shows an
emission band centered around 447 nm and corresponding
to a quantum yield of 0.040Æ0.001, which is nearly fivefold
lower than that of the free ribonucleoside (Figure 6,
Table S3 in the Supporting Information). Unlike the free ri-
bonucleoside, which exhibits monoexponential fluorescence-
intensity decay kinetics, oligoribonucleotide 8 displays biex-
ponential decay kinetics (Figure S6 and Table S3 in the Sup-
porting Information). The biexponential decay kinetics con-
sists of a shorter and a longer lifetime component, both of
which contribute equally to the overall lifetime (tav =
2.21 ns). Similar decay profiles have been reported for 2AP,
in that the free nucleoside exhibits decay kinetics corre-
sponding to a single lifetime, but if the nucleoside is incor-
porated into an oligonucleotide, it shows decay kinetics cor-
responding to multiple lifetimes.[34] The quenching of ribo-
nucleoside fluorescence in 8 may possibly be due to an elec-
tron-transfer process between the modified base and neigh-
boring bases or to alterations in the conformation of the
benzofuran moiety with respect to the nucleobase.[21,33–35]
Several emissive nucleoside analogues containing conjugat-
ed and fused heterocycles, if incorporated into oligonucleo-
tides, show similar fluorescence quenching.[6]
Figure 4. MALDI-TOF MS spectrum of modified transcript 8 calibrated
relative to the +1 ion of an internal 18-mer DNA oligonucleotide stan-
dard (m/z 5466.6). m/z 2732.6 is the +2 ion of the internal DNA stan-
dard. m/z calcd for 8: 3531.0 [M]; found: 3530.9.
The fluorescence quenching is much more pronounced in
RNA–DNA (8·9) and RNA–RNA (8·13) duplexes in which
the emissive ribonucleoside 5 is placed opposite to the com-
plementary bases, dA and A, respectively (Figure 6). Du-
Figure 5. HPLC profile of ribonucleoside products obtained from an en-
zymatic digestion of transcript 8 at l=260 nm. a) Mixture of natural ribo-
nucleosides and modified fluorescent ribonucleoside 5; b) digested oligo-
ACHTUNGTRENNUNGribonucleotide 8. See the Experimental Section for details.
Figure 6. Emission spectra (measured at 1 mm) of ribonucleoside 5, single-
stranded RNA transcript 8, and duplexes 8·9 and 8·13 in cacodylate
buffer (20 mm, pH 7.1). Samples were excited at the absorption maximum
of the ribonucleoside (l=322 nm) with an excitation slit width of 4 nm
and emission slit width of 7 nm.[25]
side analogues incorporated into oligonucleotides are
known to be influenced by a variety of mechanisms involv-
ing neighboring bases.[15,21,32,33] Therefore, steady-state and
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Fluorescent Pyrimidine Ribonucleoside Analogues
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plexes 8·9 and 8·13 show slightly blueshifted emission
maxima (approximately 439 nm), which correspond to quan-
tum yields of (0.0085Æ1.0)ꢂ10À4 and (0.0082Æ2.1)ꢂ10À4,
nearly four- and fivefold lower than that of the single-
stranded oligoribonucleotide 8, respectively (Table S3 in the
Supporting Information). The perfect duplexes also show
biexponential decay kinetics similar to that of 8 (Figure S8
in the Supporting Information). The drastic quenching of
fluorescence intensity accompanied by a small spectral shift
in perfect duplexes 8·9 and 8·13 may possibly be due to a
combination of the following reasons: a) an electron-transfer
process between the base-paired fluorescent analogue and
flanking bases, b) a desolvation effect, or c) alterations in
the conformation of the labeled uridine.[15,21,33]
Interestingly, mismatched duplexes 8·10, 8·11, and 8·12,
which contain 5 opposite to dG, dT, and dC, respectively, ex-
hibit two- to threefold higher fluorescence intensity than the
perfect duplexes (Figure S9 in the Supporting Information).
Similar to the perfect duplexes, all mismatched duplex con-
structs display biexponential decay kinetics with varying
shorter and longer lifetime components (Figure S10 and
Table S3 in the Supporting Information). Collectively, these
results demonstrate the ability of ribonucleoside 5 to photo-
physically distinguish between a single-stranded oligonucleo-
tide and a perfect complementary duplex, albeit with
quenched emission. Nevertheless, this trait of ribonucleoside
5 can be utilized in hybridization assays, in that the kinetics
of formation and dissociation of a perfect duplex can be
studied by monitoring changes in the photophysical proper-
ties of the fluorescently modified oligoribonucleotide.[18b,36]
Figure 7. Emission spectra (measured at 1 mm) of duplexes containing the
emissive ribonucleoside 5 opposite to complementary bases (8·9 and
8·13) and abasic sites (8·14 and 8·15) in cacodylate buffer (20 mm,
pH 7.1). Samples were excited at 330 nm with an excitation slit width of
7 nm and emission slit width of 9 nm.[25]
the solvent-polarity environment around the fluorophore in
8·14 and the free nucleoside is similar (Table S3 in the Sup-
porting Information). In addition, a fourfold higher kr/knr
ratio, calculated from the F and t values, indicates that the
radiative pathway is appreciably favored in the abasic-site-
containing duplex 8·14 over the perfect duplex (Table S3 in
the Supporting Information). Therefore, the emissive ribo-
nucleoside, when incorporated into an oligoribonucleotide,
preferentially signals the presence of a DNA abasic site with
a significant enhancement in fluorescence intensity.
Fluorescence detection of a DNA abasic site: Abasic sites
are frequently occurring DNA lesions that are vulnerable to
strand breaks and, if unrepaired, can be toxic as well as mu-
tagenic.[37,38] Most methods that have been developed to
detect abasic sites either use aldehyde-reactive probes,
which irreversibly react with abasic sites of isolated DNA,
or fluorescent probes, which show changes in fluorescence
properties if placed opposite to abasic sites.[12d,39,40] The dras-
tic quenching in fluorescence intensity upon placement of
the fluorescent probe 5 opposite to complementary bases
prompted us to study the effect of abasic sites on the fluo-
rescence properties of 5. Abasic-site-containing duplexes
have been constructed by hybridizing RNA transcript 8 to
custom DNA and RNA oligonucleotides 14 and 15, which
contain a chemically stable abasic-site surrogate, tetrahydro-
furan (Figure 2). When excited at 330 nm, the abasic-site-
Stability of the duplexes: The modified ribonucleoside upon
incorporation can potentially perturb the native structure of
oligoribonucleotides, which can lead to ineffective hybridiza-
tion. The observed fluorescence properties of the duplexes
would then be a consequence of a mixture of the more emis-
sive single-stranded oligoribonucleotide 8 and the corre-
sponding duplex. In order to investigate the influence of
benzofuran modification on the duplex stablity, thermal de-
naturation and radioactive native gel mobility shift experi-
ments were performed under the conditions employed in
the fluorescence experiments. Slightly lower Tm values for
the modified duplexes relative to those of the corresponding
control unmodified duplexes indicated a marginal destabili-
zation due to benzofuran modification (Figure S11 and S12
and Table S4 in the Supporting Information). 32P-labeled
transcript 8, synthesized by a transcription reaction in the
presence of template D1 and a-32P ATP, was annealed to
custom oligonucleotides 9–15. The hybridized oligonucleo-
tides were resolved on a nondenaturing polyacrylamide gel
and imaged. The gel shift experiments revealed complete
hybridization (Figure 8). Taken together, these results clear-
ly indicate that the benzofuran moiety does not affect the
hybridization efficiency. Hence, it can be concluded that the
observed differences in the fluorescence properties of com-
pletely intact duplexes are exclusively due to differences in
the microenvironment of the fluorescent ribonucleoside.
containing duplex 8·14 shows a strong emission band (lem
=
447 nm), which corresponds to a quantum yield of 0.034Æ
0.001, nearly fourfold higher than that of the perfect duplex
8·9 (Figure 7, Table S3 in the Supporting Information). Inter-
estingly, RNA–RNA duplex 8·15, which possesses an abasic
site opposite to 5, exhibits only a slight enhancement in
fluorescence intensity relative to that of the equivalent per-
fect duplex 8·13 (Figure 7, Table S3 in the Supporting Infor-
mation). The comparable emission maxima of duplex 8·14
and free ribonucleoside 5 in aqueous buffer indicates that
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S. G. Srivatsan and A. A. Tanpure
The reaction contained 2 mm GTP, CTP, ATP, and UTP or modified UTP
6, 20 mm MgCl2, 0.4 UmLÀ1 RNase inhibitor (RiboLock), 300 nm an-
nealed template, and 800 U T7 RNA polymerase. After the reaction had
been incubated for 12 h at 378C, the precipitated pyrophosphate was re-
moved by centrifugation. The reaction volume was reduced to approxi-
mately one third by Speed Vac, and loading buffer (50 mL) was added.
The solution was heated at 758C for 3 min and cooled on an ice bath.
The sample was loaded onto a preparative 20% denaturing polyacryl-
amide gel and was run at a constant power (25 W) for nearly 4.5 h. The
gel was UV shadowed, and the appropriate band was excized, extracted
with 0.3m sodium acetate, and desalted by using a Sep-Pak classic C18
cartridge. Typical yields of transcripts were 14–15 nmol. Control tran-
Figure 8. Gel mobility shift experiments to determine the hybridization
efficiency of duplexes containing the emissive ribonucleoside 5 opposite
to complementary bases, mismatched bases, and abasic sites. a) Lane 1:
single-stranded oligoribonucleotide 8; lanes 2–6: duplexes 8·9, 8·11, 8·10,
8·12, and 8·13, respectively. b) Lane 1: oligoribonucleotide 8; lanes 2–5:
duplexes 8·9, 8·14, 8·13, and 8·15, respectively.
script 7: e260 =9.10ꢂ104 mÀ1 cmÀ1
104 mÀ1 cmÀ1
; modified transcript 8: e260 =9.86ꢂ
.
Enzymatic digestion of transcript 8: Modified oligoribonucleotide 8 from
a large-scale transcription reaction (nearly 4 nmol) was digested with
snake-venom phosphodiesterase I (0.01 U), calf-intestine alkaline phos-
phatase (1 UmLÀ1), and RNase A (0.25 mg) in a total volume of 100 mL in
50 mm Tris-HCl buffer (pH 8.5, 40 mm MgCl2, 0.1 mm EDTA) for approx-
imately 12 h at 378C. After this period, RNase T1 (0.2 UmLÀ1) was
added, and the sample was incubated for another 4 h at 378C. The ribo-
nucleoside mixture obtained was analyzed by reversed-phase analytical
HPLC with a Phenomenex-Luna C18 column (250ꢂ4.6 mm, 5 mm) at l=
260 and 320 nm. Conditions: mobile phase A: 50 mm triethylammonium
acetate buffer (pH 7.5); mobile phase B: acetonitrile; flow rate:
Conclusion
Incorporation of the modified ribonucleoside reporter near
the interaction site and close maintainance of the structural
and functional integrity of the target oligoribonucleotide
will provide a better understanding of the interaction under
investigation. In this regard, microenvironment-sensitive
emissive pyrimidine ribonucleoside analogue 5, which is
minimally perturbing and displays emission in the visible
region with a reasonable quantum yield, has the potential to
be implemented in fluorescence-based assays to investigate
nucleic acid structure, dynamics, and recognition processes.
Efforts to explore therapeutically relevant RNA–protein
and RNA–small-molecule interactions by using the labeled
oligoribonucleotides are currently in progress.
1 mLminÀ1
10 min.
; gradient: 0–10% B over 20 min and 10–100% B over
Acknowledgements
We thank Prof. Sanjeev Galande for providing the facility to perform ra-
diolabeling experiments. S.G.S. is grateful to the Department of Science
and Technology, India for the financial support. A.A.T. is thankful to the
Council of Scientific and Industrial Research, India for a graduate re-
search fellowship.
Experimental Section
Enzymatic incorporation of ribonucleoside triphosphate 6:
Transcription reactions with a-32P ATP: Promoter–template duplexes
were assembled by heating a 1:1 mixture (final concentration: 5 mm) of
the deoxyoligonucleotide template (D1–D5) and an 18-mer T7 RNA
polymerase consensus promoter deoxyoligonucleotide sequence in TE
buffer (10 mm tris(hydroxymethyl)aminomethane/HCl (Tris-HCl), 1 mm
ethylenediaminetetraacetate (EDTA), 100 mm NaCl, pH 7.8) at 908C for
3 min and cooling the solution slowly to room temperature. The duplexes
were then placed over crushed ice for 20 min and stored at À408C. Tran-
scription reactions were performed in Tris-HCl buffer (40 mm, pH 7.9)
containing 250 nm annealed templates, 10 mm MgCl2, 10 mm NaCl, 10 mm
dithiothreitol (DTT), 2 mm spermidine, 1 UmLÀ1 RNase inhibitor (Ribo-
Lock), 1 mm GTP, 1 mm CTP, 1 mm UTP and/or 1 mm modified UTP 6,
20 mm ATP, 5 mCi a-32P ATP, and 3 UmLÀ1 T7 RNA polymerase in a total
volume of 20 mL. After 3.5 h at 378C, reactions were quenched by adding
loading buffer (20 mL; 7m urea in 10 mm Tris-HCl, 100 mm EDTA,
0.05% bromophenol blue, pH 8). The samples were heated at 758C for
3 min and cooled on an ice bath. The samples (4 mL) were loaded on an
analytical denaturing polyacrylamide gel (18%) containing 7m urea and
run at a constant power (11 W) for nearly 4 h. The gel was exposed to X-
ray film (1–2 h), and the exposed film was developed, fixed, and dried.
The bands corresponding to full-length transcripts were then quantified
by using software (GeneTools from Syngene) to determine the transcrip-
tion yield. The percentage incorporation of modified ribonucleoside tri-
phosphate 6 has been reported with respect to transcription efficiency in
the presence of natural NTPs. All reactions were performed in duplicate,
and the errors in yields were found to be ꢀ4%.
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Fluorescent Pyrimidine Ribonucleoside Analogues
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Published online: September 28, 2011
Chem. Eur. J. 2011, 17, 12820 – 12827
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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