A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine senses mismatched pairing

Article abstract of DOI:10.1039/b801054d

A highly emissive nucleobase analog, based on a thieno[3,4-d]pyrimidine core, is enzymatically incorporated into RNA oilgonucleotides that function as base discriminating fluorescent probes. The Royal Society of Chemistry.

Full text of DOI:10.1039/b801054d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine
senses mismatched pairing†
Seergazhi G. Srivatsan, Haim Weizman and Yitzhak Tor*
Received 21st January 2008, Accepted 22nd February 2008
First published as an Advance Article on the web 10th March 2008
DOI: 10.1039/b801054d
A highly emissive nucleobase analog, based on a thieno[3,4-
d]pyrimidine core, is enzymatically incorporated into RNA
oilgonucleotides that function as base discriminating fluores-
cent probes.
RNA strand to photophysically distinguish between perfect and
mismatched base pairing upon hybridization to DNA oligonu-
cleotides.
Short RNA constructs were selected in this case as fluorescent
hybridization probes as such oligonucleotides tend to form stable
heteroduplexes with DNA, frequently of higher stability than
the corresponding DNA–DNA duplexes.11 Although solid-phase
synthesis is a powerful tool for the site specific modification of
nucleic acids, we have chosen transcription reactions as a method
of choice for incorporating the fluorescent nucleotide into short
RNA oligonucleotides due to the following reasons: (a) the one
step preparation of the necessary triphosphate is simple, not
requiring extensive protection of the nucleoside, (b) transcription
reactions provide the desired RNA product in one step, eliminating
the lengthy deprotection steps that follow typical solid-phase
oligoribonucleotide synthesis; (c) reasonably large quantities of
RNA can be synthesized by transcription reactions with minuscule
amounts of a triphosphate; and (d) several modified constructs can
easily be made by changing the transcription template.
Thieno[3,4-d]pyrimidine 3 was synthesized in two steps from
commercially available starting materials (Scheme 1). Methyl
3-aminothiophene-4-carboxylate hydrochloride (1) was treated
with KOCN to yield the corresponding urea, which cyclized to
the corresponding pyrimidine 3 upon treatment with sodium
methoxide in methanol.12 The emissive heterocycle 3 was con-
verted into the ribonucleoside 4 using a standard glycosylation
A fundamentally simple yet practically challenging task in nucleic
acid diagnostics is the detection of single nucleotide polymorphism
(SNP).1 Such mutated base pairs, while in many cases associated
with benign biological outcomes, could also be linked to certain
diseases or reflect susceptibility to specific therapeutics.2 Whereas
several approaches for the detection of SNPs have been developed
and commercialized,3,4 efforts have recently been made to advance
base-discriminating fluorescent (BDF) nucleosides, a term coined
by Saito et al.5 Upon hybridization to a given target, such emissive
modified nucleobases can, in principle, identify the paired base
on the opposite strand by eliciting a specific change in their
photophysical characteristics.6 Such fluorescence-based tools can
facilitate the future development of enzyme-free and homogenous
SNP detection protocols. They present, however, a considerable
challenge in nucleobase probe design, as our ability to correlate
structure and microenvironment with photophysical properties is
currently limited.7
Our program aims at the development of simple and minimally
perturbing emissive nucleobases. The primary design principle
entails maintaining the highest possible structural similarity to the
natural nucleobases, while bestowing useful photophysical prop-
erties upon the probes. Of particular significance is their ability
to respond to and report on changes in their microenvironment.
Several useful nucleobase analogs have been obtained by either
conjugating or fusing five-membered aromatic heterocyclic rings
onto the pyrimidines at their 5 or 5,6 positions, respectively.8
Conjugating furan to a dU core, for example, has generated an
emissive analog that responds to the presence of abasic sites in
DNA via enhanced emission.9 The analogous furan containing
ribonucleoside has been shown to serve as a useful probe for
monitoring RNA–ligand binding.10 Here we report on a new motif
for highly emissive nucleobase analogs based on a thieno[3,4-
d]pyrimidine core. We report the synthesis of the ribonucleoside,
its basic photophysical characteristics, and its conversion into a
triphosphate, as well as its enzymatic incorporation into RNA
oligonucleotides. We then illustrate the ability of the emissive
Department of Chemistry and Biochemistry, University of California, San
Diego, La Jolla, CA, 92093-0358, USA. E-mail: ytor@ucsd.edu; Fax: 858
534 0202; Tel: 858 534 6401
† Electronic supplementary information (ESI) available: Details of syn-
thesis and characterization of compounds, transcription reactions and gel
shift experiments, and additional fluorescence and thermal melting data.
See DOI: 10.1039/b801054d
Scheme 1 Synthesis of thieno[3,4-d]pyrimidine-based emissive nucleo-
side and its triphosphate. Reagents: (a) KOCN, aq. acetic acid, RT, 81%; (b)
NaOMe, MeOH, RT; (c) (i) N,O-bis(trimethylsilyl) acetamide, TMSOTf,
CH3CN, RT, 57% (steps b and c); (ii) NH4OH, dioxane, 60 ◦C, 84%; (d) (i)
POCl3, (MeO)3PO, 0–4 ◦C; (ii) tributylammonium pyrophosphate, Bu3N,
0–4 ◦C, 56%.
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procedure13 employing 1-O-acetyl-2,3,5-tri-O-Bz-ribofuranoside
and TMSOTf, followed by deprotection with aqueous ammonia.
The triphosphate 5, required for enzymatic RNA synthesis, was
then prepared in a reasonable yield by treating 4 with POCl3
in trimethylphosphate followed by tributylammonium pyrophos-
phate (Scheme 1). Ion exchange chromatography followed by
HPLC purification provided the analytically pure triphosphate
suitable for transcription reactions.
The sensitivity of the nucleoside’s photophysical characteristics
to changes in its microenvironment was evaluated prior to its
incorporation into RNA oligonucleotides (Table 1). Nucleoside
4 displays two major absorption maxima (260 and 304 nm), which
exhibit minimal sensitivity to solvent polarity (Fig. 1). When an
aqueous solution of the nucleoside is excited at 304 nm, a strong
emission is observed at 412 nm (Fig. 1), with a quantum yield of
0.48 0.05.12,14 In contrast to the ground state absorption profiles,
the emission maximum (kem) and intensity are considerably
affected by solvent polarity (Fig. 1, Table 1). As solvent polarity
is decreased from water to methanol, and finally acetonitrile,
significant hypsochromic shift (26 nm) and hypochromic effect
(>3-fold) are observed. Excellent linear correlation between
emission energy and ET(30), Reichardt’s microscopic solvent
polarity parameter,15 is observed (Figure S1†), suggesting that the
highly emissive nucleoside is responsive to its microenvironment,
a necessary property for a useful nucleobase probe.16
incorporation into oligonucleotides.17 To preliminarily evaluate
the impact of the native nucleobases on the excited state of the new
emissive nucleoside, nucleoside monophosphates (AMP, TMP,
GMP and CMP) were titrated into a buffered solution of nu-
cleoside 4 and emission spectra were taken.12 Stern–Volmer plots
illustrate that GMP exhibits greater quenching than TMP and
AMP with quenching constants (Ksv) of 0.3, 0.1 and 0.02 mM−1,
respectively (Fig. 2). CMP displays a nonlinear behavior, but
significant quenching, comparable to that of GMP, is observed
at high concentrations.
Fig. 2 Steady-state Stern–Volmer plot for the titration of nucleoside 4
with AMP, GMP, CMP and TMP.
As noted above,10,18 enzymatic incorporation of modified nucle-
osides could be a powerful approach for the synthesis of useful
unnatural RNA constructs. To explore if the modified nucleoside
is suitable for enzymatic incorporation, transcription reactions
with T7 RNA polymerase and triphosphate 5, were performed.
A duplex DNA template was assembled by annealing the 18-
mer T7 consensus promoter to a synthetic DNA template 6
(Fig. 3).19 This template contains a unique dA residue to direct
the incorporation of the modified U analog. It also contains a
unique T residue at the 5ꢀ-end. When performed in the presence of
a-32P ATP, a successful transcription reaction would result in the
3ꢀ-end labeling of the RNA transcript, which could be resolved
on a denaturing sequencing polyacrylamide gel and visualized.
Truncated RNA transcripts would remain undetected in failed
transcription reactions. A phosphorimage of the PAGE resolved
transcription reaction with template 6, showed the formation of
full length 10-mer RNA product 7 (Fig. 4, lane 2), corresponding
Fig. 1 Absorption (100 lM) and emission (5.0 lM) spectra of nucleoside
4 in water (red), methanol (blue) and acetonitrile (green). Excitation
wavelength was 304 nm, and excitation and emission slit widths were 3
and 5 nm, respectively. Stock solutions for absorption and emission spectra
contained 10% and 0.5% DMSO, respectively.
Diverse interactions with neighboring bases can affect the
photophysical characteristics of an emissive nucleoside upon
to an efficiency of 51
6% relative to an unmodified RNA
Table 1 Photophysical data of thiophene-modified uridine 4a
ET(30)/Kcal mol−1 Energy/cm−1
c
Solvent
kmaxb/nm
kem/nm
Irel
Water
Methanol
Acetonitrile
304
304
304
412
404
386
3.3
2.9
1.0
63.10
55.40
45.60
2.43 × 104
2.48 × 104
2.59 × 104
a Conditions for absorption and emission spectra: 100 and 5.0 lM, respectively. b The lowest energy maximum is given. c Relative emission intensity with
respect to intensity in acetonitrile.
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Fig. 3 Enzymatic incorporation of thiophene-modified ribonucleoside
triphosphate 5. DNA template 6 annealed to 18mer consensus T7 promoter
and corresponding RNA transcripts (7 and 8). Also shown are synthetic
complementary and mismatch deoxyribooligonucleotides (9–12) used in
this study.
Fig. 5 HPLC profile of enzymatic digestion of transcript 7 at 260 nm.
(a) Authentic nucleoside samples and modified nucleoside 4. (b) Digested
RNA transcript. See ESI for details.†
DNA complement 9 (Fig. 6). This quenching of fluorescence
could be attributed to partial stacking of the chromophore and
alteration of its microenvironment, or by neighboring bases via
a photoinduced electron transfer mechanism, or both.22 Similar
reduction in emission has also been seen with other fluorescent
nucleoside analogs (e.g., 2-aminopurine, pyrroloC).23 Remarkably,
the emission of the duplex (7•10) containing the fluorescent
nucleoside 4 opposite to C, was enhanced over 7-fold as compared
to the perfect duplex 7•9 (Fig. 6), where 4 is found opposite to
A. Mismatched duplexes 7•11 and 7•12, placing T and G opposite
Fig. 4 Transcription reaction to study the incorporation efficiency of
thiophene-modified triphosphate 5.
transcript 8, formed in the presence of natural NTPs only (Fig. 4,
compare lanes 1 and 2). Slower migration of the modified RNA
transcript 7 compared to the unmodified transcript 8 is indicative
of the incorporation of the higher molecular weight modified
nucleoside (Fig. 4, compare lanes 1 and 2). When the transcription
reaction was performed with this template in the presence of
equimolar concentrations of UTP and 5, T7 RNA polymerase
exhibited a preference for the natural UTP (Fig. 4, lane 3). A
control reaction in the absence of 5 did not result in the formation
of any full-length product (Fig. 4, lane 4). Taken together,
these observations clearly indicate that the enzyme accepts and
incorporates the unnatural triphosphate 5 into the RNA strand
with reasonable efficiency when not in competition with UTP.20
Larger quantities of oligoribonucleotide 7 were made by large-
scale transcription reactions using template 6. MALDI-TOF MS
measurement of the PAGE purified transcript established the
integrity of the modified full-length RNA (Figure S4†).12 To
further ascertain the presence of the intact thiophene-modified nu-
cleobase in the RNA transcript, enzymatic digestions of transcript
7 with snake venom phosphodiesterase I, calf intestine alkaline
phosphatase, RNase A, and RNase T1 were performed.12 HPLC
analysis of the resulting ribonucleoside mixture unambiguously
revealed the presence of the modified nucleoside 4 and verified the
expected stoichiometry (Fig. 5).21
While the RNA transcript 7 showed an emission at 412 nm,
similar to the emission profile of the free nucleoside 4 in water, its
intensity was ∼5.5-fold lower than the free nucleoside (Figure S5†).
Further quenching was observed upon hybridization to a perfect
Fig. 6 Emission spectra of duplexes (1 lM) in 20 mM cacodylate buffer
(500 mM NaCl, 0.5 mM EDTA, pH 7.0) at 25 ◦C. See ESI for details.†
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to 4, respectively, while exhibiting stronger emission compared
to the perfect duplex 7•9, were still significantly weaker than
observed for a 4•C mismatch in 7•10. Nucleoside 4 therefore
positively responds to the presence of a C nucleoside in a
complementary oligodeoxynucleotide and can be classified as a
base discriminating fluorescent nucleoside.
In summary, the thieno[3,4-d]pyrimidine core represents a new
motif for emissive nucleobase analogs, with emission in the
visible range and respectable quantum yield. The corresponding
ribonucleoside triphosphate can be enzymatically incorporated
into RNA oligonucleotides using T7 RNA polymerase and the
resulting fluorescent constructs can be employed as hybridization
probes, positively reporting the presence of a C mismatch by
enhanced emission. Nucleobase 4 therefore expands the repertoire
of emissive isomorphic nucleoside analogs and opens up new
opportunities to explore photophysical properties of modified
nucleic acids.
Several factors could be responsible for the enhanced emission
observed for the mismatched duplex 7•10. Possibly the simplest
one would be severe destabilization caused by the putative 4-C
interaction leading to ineffective hybridization and, consequently,
high abundance of the more emissive single stranded RNA 7.
To preemptively address such a possibility, we have conducted
the hybridization and fluorescence experiments at slightly elevated
ionic strengths (500 mM NaCl). Importantly, thermal denatura-
tion studies with all 4-containing duplexes and their corresponding
control unmodified duplexes indicated that the incorporation
of the thiophene modification had minimal effect on duplex
stability (Table 2). While the perfectly-matched and least emissive
duplex 7•9 suffered a ∼6 ◦C of destabilization, all modified
duplexes were as stable as or more stable than their unmodified
counterparts (Table 2, Figures S6, S7†). Additional native gel
retardation experiments with 32P-labeled transcript RNA 7 with
ssRNA as the control (Figure S8†) showed that all duplexes were
completely intact under the condition utilized for the emission
measurements.12 Taken together, these results suggest that the
modified nucleobase is likely to reside inside the helix and that
the drastic emission enhancement observed for a mismatched
duplex where 4 is found opposite C, when compared to a perfect
duplex with 4 opposite A, is likely to result from differences in the
microenvironment of the fluorescent nucleobase.
Numerous mechanisms, operating either in the ground or
excited states, may account for the different impact of the opposite
base upon the photophysical characteristics of an emissive nucle-
obase. Bulging out of a duplex, differential hydration of a non-
canonical base pair, or base-pairing mediated tautomerization,
constitutes a few ground state processes. Photo-induced electron
or proton transfer represents plausible excited state events. Based
on the Stern–Volmer titrations reported above it is clear that
quenching by specific nucleobases is not directly correlated with
the observed enhanced emission upon mispairing of 4 with C,
although it is likely to impact the quantum yield of the emis-
sive nucleobase. Additional experiments are required to further
ascertain the fundamental causes of the observed photophysical
behavior of 4 when incorporated into oligonucleotides.
Acknowledgements
We thank the National Institutes of Health (GM 069773) for
support. We are grateful to Yun Xie and Mary Noe.
Notes and references
1 (a) The International HapMap Consortium, Nature, 2005, vol. 437,
p. 1299; (b) H. Haga, Y. Yamada, Y. Ohnishi, Y. Nakamura and T.
Tanaka, J. Hum. Genet., 2002, 47, 605; (c) A. J. Brookes, Gene, 1999,
234, 177; (d) A. Chakravarti, Nat. Genet. Suppl., 1999, 21, 56.
2 (a) T. Pastinen and T. J. Hudson, Science, 2004, 306, 647; (b) J. J.
McCarthy and R. Hilfiker, Nat. Biotechnol., 2000, 18, 505.
3 (a) Reviews: Y. Suh and C. Cantor, Mutat. Res., 2005, 573, 1; (b) B.
Sobrino, M. Brion and A. Carracedo, Forensic Sci. Int., 2005, 154,
181; (c) R. M. Twyman, Curr. Top. Med. Chem., 2004, 4, 1423; (d) K.
Nakatani, ChemBioChem, 2004, 5, 1623; (e) C. S. Carlson, T. L.
Newman and D. A. Nickerson, Curr. Opin. Chem. Biol., 2001, 5, 78;
(f) P. Y. Kwok, Annu. Rev. Genomics Hum. Genet., 2001, 2, 235.
4 (a) A. Friedrich, J. D. Hoheisel, N. Marme´ and J.-P. Knemeyer, FEBS
Lett., 2007, 581, 1644; (b) T. S. Kumar, J. Wengel and P. J. Hrdlicka,
ChemBioChem, 2007, 8, 1122; (c) E. Ergen, M. Weber, J. Jacob, A.
Herrmann and K. Mu¨llen, Chem.–Eur. J., 2006, 12, 3707; (d) U.
Asseline, M. Chassignol, Y. Aubert and V. Roig, Org. Biomol. Chem.,
2006, 4, 1949; (e) L. Valis, N. Amann and H.-A. Wagenknecht, Org.
Biomol. Chem., 2005, 3, 36; (f) K. Yamana, Y. Fukunaga, Y. Ohtani, S.
Sato, M. Nakamura, W. J. Kim, T. Akaike and A. Maruyama, Chem.
Commun., 2005, 2509; (g) A. O. Crockett and C. T. Wittwer, Anal.
Biochem., 2001, 290, 89; (h) P. L. Paris, J. M. Langenhan and E. T.
Kool, Nucleic Acids Res., 1998, 26, 3789.
5 A. Okamoto, Y. Saito and I. Saito, Photochem. Photobiol. C: Pho-
tochem. Rev., 2005, 6, 108.
6 (a) K. Tainaka, K. Tanaka, S. Ikeda, K.-I. Nishiza, T. Unzai, Y.
Fujiwara, I. Saito and A. Okamoto, J. Am. Chem. Soc., 2007, 129,
4776; (b) Y. Saito, E. Mizuno, S. S. Bag, I. Suzuka and I. Saito, Chem.
Commun., 2007, 4492; (c) F. Takei, H. Suda, M. Hagihara, J. Zhang, A.
Kobori and K. Nakatani, Chem.–Eur. J., 2007, 13, 4452; (d) R. H. E.
Hudson and A. Ghorbani-Choghamarani, Org. Biomol. Chem., 2007,
5, 1845; (e) J. H. Ryu, Y. J. Seo, T. Hwang, J. Y. Lee and B. H. Kim,
Tetrahedron, 2007, 63, 3538; (f) Q. Xiao, R. T. Ranasinghe, A. M. P.
Tang and T. Brown, Tetrahedron, 2007, 63, 3483; (g) N. Moran, D. M.
Bassani, J.-P. Desvergne, S. Keiper, P. A. S. Lowden, J. S. Vyle and
J. H. R. Tucker, Chem. Commun., 2006, 5003; (h) E. Mayer-Enthart and
H.-A. Wegenknecht, Angew. Chem., Int. Ed., 2006, 45, 3372; (i) D. J.
Hurley, S. E. Seaman, J. C. Mazura and Y. Tor, Org. Lett., 2002, 4,
2305.
Table 2 Thermal melting of duplexes derived from modified (7) and
unmodified (8) RNA transcriptsa
Tm/◦C
Tm/◦C
7 Y. Tor, Tetrahedron, 2007, 63, 3425.
8 (a) N. J. Greco and Y. Tor, Tetrahedron, 2007, 63, 3515; (b) Y. Tor,
S. Del Valle, D. Jaramillo, S. G. Srivatsan, A. Rios and H. Weizman,
Tetrahedron, 2007, 63, 3608.
9 N. J. Greco and Y. Tor, J. Am. Chem. Soc., 2005, 127, 10784.
10 (a) S. G. Srivatsan and Y. Tor, J. Am. Chem. Soc., 2007, 129, 2044;
(b) S. G. Srivatsan and Y. Tor, Tetrahedron, 2007, 63, 3601.
11 (a) S. Wang and E. T. Kool, Biochemistry, 1995, 34, 4125; (b) N.
Sugimoto, S. Nakano, M. Yoneyama and K. Honda, Nucleic Acids
Res., 1996, 24, 4501; (c) F. Barone, L. Cellai, M. Matzeu, F. Mazzei
and F. Pedone, Biophys. Chem., 2000, 86, 37; (d) H. Binder, J. Phys.:
Condens. Matter, 2006, 18, S491.
Modified duplex
Unmodified duplex
7•9
53.3 0.7
52.8 0.1
54.6 0.5
50.3 0.7
8•9
59.4 0.1
48.3 0.7
50.3 0.7
49.8 1.4
7•10
7•11
7•12
8•10
8•11
8•12
a Duplexes were formed by annealing a 1 : 1 mixture of the oligonucleotides
in 20 mM cacodylate buffer (pH 7.0, 500 mM NaCl, 0.5 mM EDTA).
Concentration of each duplex was 1 lM.12
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12 See ESI†.
20 Experiments with additional templates (see ESI, Figure S2 and S3†)
illustrate that T7 RNA polymerase cannot introduce the modified
nucleoside at positions that are closer to the promoter.
13 F. Seela and X. Peng, J. Org. Chem., 2006, 71, 81.
14 Determined relative to 2-aminopurine.
15 C. Reichardt, Chem. Rev., 1994, 94, 2319.
21 Mass spectral analysis of individual HPLC fractions further confirmed
the authenticity of the native and modified nucleosides present in the
enzymatic digest.
22 M. Torimura, S. Kurata, K. Yamada, T. Yokomaku, Y. Kamagata, T.
Kanagawa and R. Kurane, Anal. Sci., 2001, 17, 155.
23 (a) J. M. Jean and K. B. Hall, Proc. Natl. Acad. Sci. USA, 2001, 98, 37;
(b) C. Liu and C. T. Martin, J. Mol. Biol., 2001, 308, 465.
16 R. W. Sinkeldam and Y. Tor, Org. Biomol. Chem., 2007, 5, 2523.
17 (a) E. L. Rachofsky, R. Osman and J. B. A. Ross, Biochemistry, 2001,
40, 946; (b) D.-G. Xu and T. M. Nordlund, Biophys. J., 2000, 78,
1042.
18 S. G. Srivatsan and Y. Tor, Nature Protocols, 2007, 2, 1547.
19 Y. Tor and P. B. Dervan, J. Am. Chem. Soc., 1993, 115, 4461.
1338 | Org. Biomol. Chem., 2008, 6, 1334–1338
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