Synthesis, photophysical properties and incorporation of a highly emissive and environment-sensitive uridine analogue based on the lucifer chromophore

Article abstract of DOI:10.1002/cbic.201402052

The majority of fluorescent nucleoside analogues used in nucleic acid studies have excitation maxima in the UV region and show very low fluorescence within oligonucleotides (ONs); hence, they cannot be utilised with certain fluorescence methods and for cell-based analysis. Here, we describe the synthesis, photophysical properties and incorporation of a highly emissive and environment-sensitive uridine analogue, derived by attaching a Lucifer chromophore (1,8-naphthalimide core) at the 5-position of uracil. The emissive nucleoside displays excitation and emission maxima in the visible region and exhibits high quantum yield. Importantly, when incorporated into ON duplexes it retains appreciable fluorescence efficiency and is sensitive to the neighbouring base environment. Notably, the nucleoside signals the presence of purine repeats in ON duplexes with an enhancement in fluorescence intensity, a property rarely displayed by other nucleoside analogues. Green fluorescent nucleoside: A highly emissive uridine analogue containing the Lucifer chromophore at the 5-position of uracil was incorporated into oligonucleotides. It retains appreciable fluorescence and is sensitive to the neighbouring base environment, both its adjacent bases and its partner on the complementary strand in duplexes.

Full text of DOI:10.1002/cbic.201402052

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DOI: 10.1002/cbic.201402052
Synthesis, Photophysical Properties and Incorporation of
a Highly Emissive and Environment-Sensitive Uridine
Analogue Based on the Lucifer Chromophore
Arun A. Tanpure and Seergazhi G. Srivatsan*[a]
The majority of fluorescent nucleoside analogues used in nu-
cleic acid studies have excitation maxima in the UV region and
show very low fluorescence within oligonucleotides (ONs);
hence, they cannot be utilised with certain fluorescence meth-
ods and for cell-based analysis. Here, we describe the synthe-
sis, photophysical properties and incorporation of a highly
emissive and environment-sensitive uridine analogue, derived
by attaching a Lucifer chromophore (1,8-naphthalimide core)
at the 5-position of uracil. The emissive nucleoside displays ex-
citation and emission maxima in the visible region and exhibits
high quantum yield. Importantly, when incorporated into ON
duplexes it retains appreciable fluorescence efficiency and is
sensitive to the neighbouring base environment. Notably, the
nucleoside signals the presence of purine repeats in ON du-
plexes with an enhancement in fluorescence intensity, a proper-
ty rarely displayed by other nucleoside analogues.
Introduction
Base-modified fluorescent nucleoside probes that photophysi-
cally report subtle changes in their neighbouring base environ-
ment have been very useful in designing bioanalytical assays
to study the structure and function of nucleic acids.[1,2] As nat-
ural nucleobases are essentially nonemissive, useful fluores-
cence properties have been bestowed by attaching known flu-
orophores, heterocycles or aromatic rings onto the bases.[2,3]
Fluorescent nucleosides have also been developed by using
fluorescent heterocycles (e.g., pteridines) and polycyclic aro-
matic hydrocarbons (PAHs) as base surrogates.[4,5] The fluores-
cence properties of many of these nucleoside analogues, when
incorporated into oligonucleotide (ON) sequences, are influ-
enced by collisional, H-bonding, and stacking interactions with
adjacent bases,[6] electron transfer between the fluorescent
base and neighbouring bases,[7] solvation/desolvation effects[6]
and the conformation of the fluorescent base within the ON.[8]
Over the years several such responsive fluorescent nucleoside
analogue probes have been implemented in assays, 1) to
detect base-pair mismatches,[9] abasic sites,[10] mutagenic nucle-
obase modifications[11] and electron transfer processes,[12] and
2) to study nucleic acid topologies,[13] enzyme activities[14] and
nucleic acid–small molecule and nucleic acid–protein com-
plexes.[15] However, barring a very few examples, emissive nu-
cleoside analogues, when incorporated into ON sequences, ex-
perience drastic fluorescence quenching because of interac-
tions with neighbouring bases, thus considerably limiting their
application.[16] Notably, purines (especially guanine, G) quench
the fluorescence of most fluorophores by a photoinduced elec-
tron-transfer process; hence, turn-on fluorescence detection of
purine repeats and mismatches has been quite a challenge.[17–19]
Therefore, much of the recent effort in the development of
new fluorescent nucleoside analogues is directed towards de-
signing environment-sensitive analogues that have excitation
and emission maximum in the visible region and high fluores-
cence efficiency within ONs, with the view of implementing
them in both in vitro and in vivo assays.[5,20–23]
Kool and co-workers have assembled a library of DNA-like
chains containing different PAH fluorophores (“oligodeoxy-
fluorosides”) that display large Stokes shifts and a wide array
of quantum yields and emission wavelengths.[21] These oligo-
deoxyfluorosides with tunable photophysical properties have
been implemented in the detection of molecular species in so-
lution and in the multiplexed imaging of cells. In a somewhat
similar approach, siRNAs labelled with multiple phenylpyrrolo-
cytidine residues have been used to monitor (by fluorescence
microscopy) the trafficking and silencing activity of siRNA
inside living cells.[22] More recently, a quadracyclic adenine ana-
logue and 4-aminophthalimide C-nucleoside have been intro-
duced as fluorescent nucleoside surrogates; when incorporat-
ed into ONs these form stable duplexes and retain reasonable
fluorescence efficiency, compared to most other fluorescent
nucleoside analogues.[23] As a part of the continued efforts to
develop new fluorescent probes with useful properties, we
report the synthesis and photophysical properties of a highly
emissive ribonucleoside analogue, obtained by conjugating
the Lucifer chromophore (naphthalimide core) at the 5-posi-
tion of uridine by an ethynyl linker.[24] The naphthalimide-modi-
fied uridine analogue has excitation and emission maxima in
the visible region and exhibits excellent fluorescence solvato-
chromism. Notably, the fluorescence properties of the emissive
nucleoside incorporated into ONs is sensitive to the flanking
[a] A. A. Tanpure, Dr. S. G. Srivatsan
Department of Chemistry
Indian Institute of Science Education and Research, Pune
Dr. Homi Bhabha Road, Pashan, Pune 411008 (India)
E-mail: srivatsan@iiserpune.ac.in
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402052.
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bases and base-pair mismatches. In particular, the
emissive nucleoside signals the presence of purine
repeats (dG and dA) with a significant increase in
fluorescence intensity, a property seldom exhibited
by most of the fluorescent nucleoside analogues.[17]
Results and Discussion
Synthesis and photophysical properties of
naphthalimide-modified uridine 6
The fluorescent probe 6 is based on the 4-amino-1,8-
naphthalimide core of Lucifer dyes; this absorbs light
in the visible region and exhibits a large Stoke shift
and high quantum yield.[25,26] The photophysical
properties of the parent naphthalimide, containing
the electron-withdrawing imide moiety, largely
depend on the substituent at the 4-position of the
dye.[27] As the fluorescence properties of the naph-
thalimide core can be tuned by rational modification,
its derivatives have been extensively used as biologi-
cal markers, sensors and electroluminescent materi-
als, to name a few applications.[26] Encouraged by
these reports, it was postulated that coupling 4-eth-
ynyl-1,8-naphthalimide at the 5-position of uridine
would impart superior probe-like properties (similar
to Lucifer dyes) to the nucleobase. Naphthalimide-
modified ribonucleoside 6 was synthesised according
to the steps shown in Scheme 1. First, 4-ethynyl-1,8-
naphthalimide derivative 4 was prepared by reacting
commercially available 4-bromo-1,8-naphthalic anhy-
dride (1) with amine-modified tri(ethylene glycol)
Scheme 1. Synthesis of fluorescent uridine analogue 6 and its corresponding triphos-
phate 7.[28] a) ethanol, reflux, 90%; b) TMS-acetylene, Pd(PPh3)4, CuI, iPr2NEt, THF, RT, 87%;
c) 1m TBAF, MeOH, 608C, 66%; d) 5-iodouridine (5), Pd(PPh3)4, CuI, Et3N, DMF, RT, 85%;
e) POCl3, (MeO)3PO, ~48C; f) bis-tributylammonium pyrophosphate, Bu3N, ~48C, 31%.
linker 2, followed by a Sonogashira cross-coupling reaction
with TMS-acetylene. The silyl protecting group was then de-
protected in the presence of TBAF to afford 4 in moderate
yield. A tri(ethylene glycol) linker was attached at the imide
position to enhance the solubility of the naphthalimide deriva-
tive in polar solvents for subsequent manipulation. The fluores-
cent uridine analogue 6 was then obtained by palladium-cata-
lysed cross coupling between the 4-alkyne-modified naphthal-
imide (4) and 5-iodouridine (5).
ic solvent polarity parameter) further indicated the sensitivity
of the nucleoside to microenvironment changes (Figure S2).
The high fluorescence efficiency, absorption and emission pro-
files in the visible region and sensitivity to polarity prompted
us to study the fluorescence properties of the emissive nucleo-
side within ONs.
The ground-state and excited-state electronic properties of
nucleoside 6 were evaluated in solvents of different polarities
to test the solvatochromic behaviour of the nucleoside. The
ground-state electronic spectrum was marginally affected by
changes in solvent polarity, however, the excited-state proper-
ties were significantly altered (Figure 1, Table 1). In dioxane
(the least-polar solvent used in this study) the nucleoside
exhibited very strong emission (lem =480 nm, quantum yield
87%; Figure 1, Table 1). As the solvent polarity was increased
(from dioxane to water) the nucleoside showed a remarkable
48 nm red-shift in the emission band (lem =528) and nearly 11-
fold quenching of fluorescence intensity. Excited-state lifetime
measurements revealed longer lifetimes in nonpolar solvents
than in polar solvents (see Figure S1 in the Supporting Infor-
mation and Table 1). A good positive correlation between
Stokes shift in different solvents and ET(30) value (a microscop-
Figure 1. Absorption (25 mm, solid lines) and emission (5.0 mm, dashed lines)
spectra of nucleoside 6 in solvents of different polarity. Solutions contained
2.5 and 0.5% DMSO, respectively.[29]
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When transcription was performed with template T1, the
RNA polymerase incorporated the monophosphate of 7 at the
+7-position to afford the full-length transcript 9 in a good
yield (Figure 3, lane 2).[36] The incorporation of this heavier ribo-
nucleotide into the transcript was also evident from the retard-
ed mobility exhibited by transcript 9 (Figure 3, compare lanes
Table 1. Photophysical properties of nucleoside 6.[28,29]
[b]
[c]
Solvent
lmax[a] [nm]
lem [nm]
Irel
F[c]
tave [ns]
water
391
393
393
393
528
527
500
480
1
3.7
9.6
0.11
0.24
0.67
0.87
1.03
1.66
3.68
2.78
methanol
acetonitrile
dioxane
10.8
[a] Lowest energy maximum. [b] Fluorescence intensity is given relative
to that in water. [c] Errors for F and tave are ꢀ0.003 and ꢀ0.03 ns, re-
spectively.
Enzymatic incorporation of nucleoside 6 into RNA ONs
Primer extension and in vitro transcription reactions in the
presence of DNA and RNA polymerases, respectively, and liga-
tion reactions have been effectively used in the synthesis of
fluorescently modified DNA and RNA ONs.[15,30–32] Notably,
Hirao and co-workers have elegantly used unnatural base pairs
that are orthogonal to A–T/U and G–C to site-specifically incor-
porate fluorescent analogues of unnatural bases into RNA oli-
gonucleotides by in vitro transcription.[15a,33] Thus, we chose to
incorporate naphthalimide-modified uridine into RNA ONs by
in vitro transcription. The modified UTP 7 was synthesised by
treating 6 with anhydrous POCl3 and then with bis-tributylam-
monium pyrophosphate at ~48C (Scheme 1).[34] The efficiency
of T7 RNA polymerase to incorporate 7 into RNA ONs was first
tested by radiolabelling experiments with a series of promoter/
template DNA duplexes. The duplexes were formed by anneal-
ing a T7 RNA polymerase consensus promoter DNA ON with
template ONs T1–T5 (Figure 2).[35] The templates were de-
signed to direct the incorporation of the monophosphate of 7
at one or two positions near the promoter region, away from
the promoter region or at the 3’-end of the transcript. In vitro
transcription reactions were performed in the presence of GTP,
CTP, [a-32P]ATP and UTP/7, and the radiolabelled transcription
products were analysed by polyacrylamide gel electrophoresis
under denaturing conditions and phosphorimaged.
Figure 3. Phosphorimage of transcription products resolved by denaturing
PAGE. Transcription was performed with templates T1–T5 and UTP and/or
modified UTP (7).[29] Incorporation of 7 is given as percent relative to control
(natural UTP). Modified full-length transcripts (arrow heads) were determined
by mass analysis (Table S1). Incorporation of UTP 7 with T2 was very low;
hence, the band corresponding to the full-length transcript (10) could not
be assigned (lane 6).
1 and 2). A control reaction (absence of UTP or 7)
produced no full-length oligoribonucleotide product,
thus ruling out misincorporation (Figure 3, lane 3);
reaction in the presence of equimolar UTP and 7 in-
dicated preference for UTP over 7 (Figure 3, lane 4).
Attempts to introduce the modification near the pro-
moter region (+3 and +4, templates T2 and T3)
resulted in very low yields of full-length transcript
(Figure 3, lanes 6 and 8). This was not unexpected, as
the enzyme is often less tolerant of modifications
during the initial phase of polymerisation.[32b] Interest-
ingly, transcription with T4 results in double
incorporation (adjacent positions) with a reasonable
efficiency (Figure 3, lane 10); with template T5 the
RNA polymerase incorporated the modification at the
3’-end of the transcript (13) with excellent efficiency
(Figure 3, lane 12 and Figure S3). Although, internally
Figure 2. Incorporation of UTP 7 into RNA ONs by in vitro transcription with templates
T1–T7.[29]
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cided to examine the effect of flanking bases and mismatched
base pairs on the fluorescence properties of 6. RNA ONs 9, 14
and 15 (containing 6 between rG, rA and rC residues, respec-
tively; “rG”=G residues at each side) were synthesised by
large-scale transcription reactions (Figure 2, Figure S4,
Table S1).[29] Enzymatic synthesis of an RNA ON with 6 between
U residues was not attempted, as it would be difficult to con-
trol the site of incorporation in in vitro transcription. In addi-
tion, RNA polymerase prefers natural UTP over 7, thus prevent-
ing the introduction of 6 between U residues by transcription
reaction (Figure 3, lane 4). Therefore, we synthesised RNA
ON 20 (containing 6 in between U residues) by a solid-phase
ON synthesis protocol. The 2’-O-(tert-butyldimethylsilyl)-pro-
tected phosphoramidite substrate 19 was synthesised as
shown in Scheme 2. The modified phosphoramidite substrate
was then site-specifically incorporated into an RNA ON (se-
quence 20) by using a standard RNA ON synthesis cycle.[29] The
ON was deprotected and purified, and the presence of the
fluorescent uridine analogue in the full-length product was
confirmed by mass analysis (Figure S5, Table S1).
Figure 4. HPLC chromatogram of ribonucleoside products obtained from an
enzymatic digestion of transcript 9 at 260 nm.[29] A) Mixture of natural ribo-
nucleosides and modified fluorescent ribonucleoside 6. B) Digested ON 9.
The fractions corresponding to individual ribonucleoside fractions were fur-
ther analysed by mass spectroscopy (see Table S2).
RNA ONs 9 and 14 (6 flanked by purine residues, rG and rA,
respectively) displayed significantly quenched and slightly
blue-shifted emission bands, as compared to the free nucleo-
side. Whereas ON 20 (6 between rU residues) exhibited a dis-
modified transcript 9 and 3’-end modified transcript
13 have same mass, their electrophoretic mobilities
were different (Figure 3, compare lanes 2 and 12).
Predicting the electrophoretic mobility of modified
oligonucleotides based on charge and mass is not
straightforward, as the hydrophobicity, hydration,
conformation and frictional properties of the append-
ed group (as well as its interaction with medium) can
affect mobility.[37] Therefore, the observed difference
in mobility could be attributable to differences in the
hydrophobicity, conformation and frictional proper-
ties between the internal and 3’-end-attached fluoro-
phore.
RNA ONs were isolated from large-scale transcrip-
tion reactions to further examine the incorporation
of the naphthalimide modification into transcripts.
Mass analysis confirmed the formation of modified
full-length RNA oligonucleotides by in vitro transcrip-
tion with the fluorescent UTP (Table S1). The pres-
ence of 6 in the transcript was also established by
enzymatic digestion. RNA ON 9 was subjected to di-
gestion by phosphodiesterase, alkaline phosphatase,
RNase A and RNase T1. HPLC analysis of the resulting
ribonucleoside products revealed the presence of
modified nucleoside in the transcript (Figure 4,
Table S2).
Photophysical characterisation of naphthalimide-
modified RNA ONs
Interactions with neighbouring bases can affect the
photophysical properties of an emissive nucleoside in
Scheme 2. Synthesis of naphthalimide-modified uridine phosphoramidite substrate 19
used in the solid-phase synthesis of RNA ON 20.[28,29] DMT, 4,4’-dimethoxytrityl; TBDMS,
2’-O-tert-butyldimethylsilyl. a) 4, Pd(PPh3)4, CuI, iPr2NEt, DMF, RT, 85%; b) AgNO3, TBDMS-
ONs, by various mechanisms.[6–8,17] Such fluorescent
nucleoside analogues are very useful in designing nu-
cleic-acid-based diagnostic tools.[1,2] Therefore, we de- Cl, pyridine, THF, RT, 73%; c) iPr2NP(Cl)OEtCN, iPr2NEt, CH2Cl2, RT, 70%.
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cernible reduction in fluorescence intensity, ON 15 (6 in be-
tween rC residues) displayed a significant enhancement, com-
pared to the free nucleoside (Figure 5). The effect of neigh-
bouring bases on the fluorescence properties of 6 was further
evaluated with duplexes of ONs 9, 14, 15 and 20. The duplexes
were assembled by hybridising fluorescent ONs with comple-
mentary DNA ONs, such that the emissive nucleoside was op-
posite to complementary or mismatched bases (Figure 6).
The naphthalimide modification can potentially affect the
hybridisation efficiency of the RNA ONs, and hence, the ob-
served fluorescence profile might reflect a combination of
intact duplexes and unhybridised fluorescent ON. Therefore,
prior to performing the fluorescence study, the effect of modi-
fication on the stability of duplexes was determined by ther-
mal denaturation experiments. Although, the modification had
a slight destabilising effect, the naphthalimide-modified ONs
formed stable duplexes, with Tm values well above room tem-
perature (Figure S6, Table S3). Hence, destabilisation due to the
presence of the modification should not compromise photo-
physical characterisation of duplexes at room temperature.
When placed opposite complementary or mismatched bases
in RNA·DNA heteroduplexes (9·9A, 9·9T, 9·9G and 9·9C), 6
flanked by rG residues displayed further quenching in fluores-
cence intensity, but with no change in emission maximum rela-
tive to single stranded RNA ON 9 (Figure 7A). ON 14 duplexes
(6 flanked by rA and opposite dA, dT or dC bases) showed
enhanced fluorescence relative to 14, but their overall fluores-
cence efficiencies were weak (Figure 7B). The fluorescence
quenching exhibited for 6 upon incorporation into oligonu-
cleotide duplexes is a common feature shown by most other
nucleoside analogues. This quenching might be due to elec-
tron transfer between the modified base and neighbouring
bases.[7] However, partial stacking interaction and relative con-
formation of the naphthalimide core with respect to the uracil
base in different base environment could also influence the
fluorescence properties.[6,8]
Figure 5. Emission spectra of nucleoside 6 (1 mm) and RNA ONs 9, 14, 15
and 20 (1 mm) containing 6 between rG, rA, rC and rU residues, respectively.
ONs were excited at 407 nm; excitation and emission slit widths: 6 and
8 nm, respectively.[29]
RNA ON 15 (6 between rC residues) showed an intense
band with an emission maximum at 515 nm (Figure 8). Surpris-
ingly, the emissive uridine analogue in duplex 15·15G (consec-
utive dG residues, 6 opposite dG) displayed more than twofold
higher fluorescence intensity, relative to when it was opposite
its complementary base (dA, duplex 15·15A; Figure 8). Howev-
er, the fluorescence intensity was not significantly higher when
the nucleoside analogue was opposite dT or dC (mismatches
in 15·15T and 15·15C, respectively). We were doubly surprised
when the fluorescent uridine analogue was incorporated be-
tween rU residues and opposite complementary (20·20A) and
Figure 6. Sequences of custom DNA ONs. Hybridisation of 9 with 9A, 9T, 9G
and 9C places the fluorescent nucleoside 6 opposite complementary base
dA and mismatched bases dT, dG and dC, respectively.
Figure 7. A) Emission spectra of ON 9 (1 mm) and duplexes of 9. B) Emission spectra of ON 14 (1 mm) and duplexes of 14. ONs were excited at 407 nm; excita-
tion and emission slit widths: 6 and 8 nm, respectively.[29]
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majority of fluorophores is quenched by purine residues,[17] the
enhancement in fluorescence intensity exhibited by naphthal-
imide-modified nucleoside in G-rich and A-rich environments is
rare and useful.[19] An analogue probe with excitation and
emission maxima in the visible region as well as the ability to
exhibit enhanced fluorescence efficiency in particular sequence
contexts could be highly useful in hybridisation assays to
detect nucleobase repeats in nucleic acids (e.g., G-repeats and
A-repeats).
Conclusions
Nucleic acids interact with proteins, nucleic acids and small-
molecule metabolites; during such recognition events they un-
dergo conformational changes both at the global and nucleo-
side levels.[38] The changes in nucleoside conformation (e.g.,
near the protein/ligand binding site) also alter its surrounding
physical properties and interactions with adjacent nucleosides.
Hence, environment-sensitive fluorescent nucleoside analogues
with excitation/emission maxima in the visible region and high
quantum yields when within ONs are suitable for both in vitro
and cell-based applications, and are highly desirable. In this
regard, fluorescent ribonucleoside 6, based on the Lucifer
chromophore, represents a new type of environment-sensitive
nucleoside analogue. The triphosphate and phosphoramidite
derivative of the naphthalimide-modified nucleoside act as
good substrates for the synthesis of fluorescent RNA ONs by in
vitro transcription and by solid-phase synthesis, respectively.
Furthermore, the emissive nucleoside incorporated into ON
duplexes exhibits appreciable fluorescence efficiency, and is
responsive to its flanking bases and base-pair substitutions.
These favourable photophysical properties and amenability to
enzymatic and chemical incorporation underscore the poten-
tial of the naphthalimide-modified nucleoside analogue as
a probe in the investigation of nucleic acids by fluorescence
spectroscopy and microscopy.[39] We are currently evaluating
the suitability of the fluorescent nucleoside analogue in prob-
ing the cellular uptake and trafficking of RNA ONs (e.g., siRNA)
by fluorescence microscopy, and the results will be reported in
due course.
Figure 8. Emission spectra of ON 15 (1 mm) and duplexes of 15. ONs were
excited at 407 nm; excitation and emission slit widths: 6 and 8 nm, respec-
tively.[29]
mismatched bases (20·20T, 20·20G, 20·20C): significantly
higher (more than fourfold) fluorescence intensity, with no ap-
parent change in emission maximum relative to single-strand-
ed ON 20 (Figure 9). This is noteworthy, because, upon incor-
poration into ONs, the majority of fluorescent nucleoside ana-
logues (including 2-aminopurine) experience drastic quenching
in fluorescence intensity due to stacking interactions and or
electron transfer.[2,6,17] In particular, this effect is more pro-
nounced when the nucleoside analogue is in the vicinity of
purines bases.[17]
Experimental Section
Figure 9. Emission spectra of ON 20 (1 mm) and duplexes of 20. ONs were
excited at 407 nm; excitation and emission slit widths: 6 and 8 nm, respec-
tively.[29]
Photophysical characterisation of ribonucleoside analogue 6
Steady-state fluorescence of ribonucleoside analogue in various sol-
vents: Analogue 6 (5 mm) in water, methanol, acetonitrile or diox-
ane was excited at the respective lowest energy absorption maxi-
mum, with excitation and emission slit widths of 1 and 5 nm, re-
spectively. Fluorescence experiments were performed in triplicate
in a micro fluorescence cell (path length 1.0 cm; Hellma Analytics,
Mꢁllheim, Germany) on a Fluorolog-3 spectrophluorometer (Horiba
Jobin Yvon, Kyoto, Japan).
The fluorescence profile exhibited by 6 in different nucleo-
base environments is likely attributable to alterations in the rel-
ative conformation of the naphthalimide moiety with respect
to the uracil base, electron transfer process between the emis-
sive nucleobase and neighbouring bases and solvation/desol-
vation effects.[6–8,17] Together, these results clearly indicate that
the fluorescence properties of the emissive base are sensitive
to flanking bases and base-pair substitutions; essentially, these
arise from changes in interactions between the modified base
and neighbouring bases. The fact that the fluorescence of the
Time-resolved fluorescence measurements: Excited-state lifetimes of
6 in various solvents were determined by using a TCSPC fluores-
cence spectrophotometer (Horiba Jobin Yvon). Ribonucleoside 6
(5 mm) was excited with a 371 nm NanoLED-371L LED source (IBH/
Horiba, Glasgow, UK). Lifetime measurements were performed in
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duplicate, and decay profiles were analysed with DAS6 analysis
software (IBH). Fluorescence intensity decay kinetics in all solvents
were monoexponential, with c2 (goodness of fit) values very close
to unity.
phoramidites was performed with a coupling time of 10 min; in-
corporation of fluorescent 2’-O-TBDMS-protected phosphoramidite
substrate 19 was performed with a coupling time of 30 min (cou-
pling efficiency 20%, based on a trityl monitor assay). The trityl
protecting group was deprotected on the synthesiser. The solid
support was treated with a solution of methylamine (10m in etha-
nol) and water (1:1, 1.5 mL) for 12 h. The mixture was centrifuged,
and the supernatant was evaporated to dryness in a SpeedVac. The
residue was then dissolved in DMSO (100 mL) and treated with
TEA·3HF (150 mL). The sample was heated at 658C for 2.5 h and
was brought to RT. The completely deprotected ON solution was
lyophilised and then purified by 20% denaturing PAGE. The band
corresponding to the full-length product was identified by UV
shadowing. The ON was extracted with ammonium acetate buffer
(0.5m, 3 mL) and desalted in a Sep-Pak classic C18 cartridge (e260
and MALDI-MS data in Table S1; ON 20 HPLC chromatogram in Fig-
ure S5).
Enzymatic incorporation of modified triphosphate 7 into RNA
ONs
Transcription reactions in presence of [a-32P]ATP: Promoter/template
duplexes were constructed by heating equimolar (5 mm) DNA tem-
plate (T1–T5) and an 18-mer T7 RNA polymerase consensus pro-
moter DNA sequence in TE buffer (Tris·HCl (10 mm, pH 7.8), EDTA
(1 mm), NaCl (100 mm)) at 908C for 3 min, and cooling slowly to
room temperature. The duplexes were placed in an ice bath for
20 min and stored at À408C. Transcription reactions were per-
formed in Tris·HCl (40 mm, pH 7.9) containing annealed template
(250 nm), MgCl2 (10 mm), NaCl (10 mm), dithiothreitol (10 mm),
spermidine (2 mm), RiboLock RNase inhibitor (1 UmLÀ1; Thermo Sci-
entific), GTP (1 mm), CTP (1 mm), UTP (1 mm) and/or modified
UTP 7 (1 mm), ATP (20 mm), [a-32P]ATP (5 mCi, 2.5 pmol) and T7
RNA polymerase (3 UmLÀ1) in a total volume of 20 mL for 4 h at
378C. Reactions were quenched by adding loading buffer (20 mL;
Tris·HCl (10 mm, pH 8), urea (7m), EDTA (100 mm), Bromophenol
Blue (0.05%)), heated (758C, 3 min) then cooled in an ice bath.
Samples (5 mL) were loaded onto an 18% denaturing polyacryl-
amide gel. The gel was exposed to X-ray film (10 min), and the ex-
posed film was developed, fixed and dried. The bands were then
quantified by using the software GeneTools (Syngene, Cambridge,
UK) to determine percentage incorporation of 7 relative to tran-
scription efficiency with natural UTP. All reactions were performed
in duplicate, and the standard deviations were found to be Æ3%.
Photophysical characterisation of naphthalimide-modified ONs:
ONs 9, 14, 15 and 20 (10 mm) were annealed to respective comple-
mentary custom DNA ONs by heating a mixture (1:1.1) of ONs in
cacodylate buffer (20 mm, pH 7.0) with NaCl (500 mm) and EDTA
(0.5 mm) at 908C for 3 min. Samples were then cooled slowly to RT,
then placed in crushed ice for 2 h. Samples were diluted (final
1 mm, with respect to 9, 14, 15 and 20) in cacodylate buffer. Fluo-
rescently modified duplexes were excited at 407 nm (excitation
and emission slit widths, 6 and 8 nm, respectively). Fluorescence
experiments were performed in triplicate in a micro fluorescence
cuvette (path length 1.0 cm, Hellma) on a Horiba Jobin Yvon, Fluo-
rolog-3 at 208C.
Large-scale transcription reactions: Large-scale transcription reac-
tions with templates T1 and T3–T7 were performed as above but
in 250 mL reaction mixtures, to isolate ONs for further characterisa-
tion and photophysical studies. GTP, CTP, ATP, UTP or 7 (2 mm
each), MgCl2 (20 mm), RiboLock (0.4 UmLÀ1), annealed template
(300 nm) and T7 RNA polymerase (800 U) were mixed and incubat-
ed for 12 h at 378C. The reaction volume was reduced (to ~1/3) in
a SpeedVac, then loading buffer (30 mL) was added. The samples
were loaded onto a preparative 20% denaturing polyacrylamide
gel and electrophoresed at a constant 24 W (~800 V) for 5 h. The
gel was UV shadowed, then the appropriate band was removed,
extracted with sodium acetate (0.3m) and desalted in a Sep-Pak
classic C18 cartridge (for purity see Figure S4). Typical transcript
yield: 16–19 nmol.
Acknowledgements
S.G.S. is grateful to CSIR, India (02-0086/12/EMR-II) for a research
grant. A.A.T. thanks CSIR, India for a graduate research fellow-
ship.
Keywords: emission spectroscopy · fluorescent nucleoside · G-
repeats · nucleoside analogues · RNA
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Received: February 21, 2014
Published online on May 23, 2014
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ChemBioChem 2014, 15, 1309 – 1316 1316