Fluorescent nucleoside analogue displays enhanced emission upon pairing with guanine

Article abstract of DOI:10.1039/c0ob00413h

A fluorescent nucleobase analogue, 7-aminoquinazoline-2,4-(1H,3H)-dione, is incorporated into a DNA oligonucleotide and senses mismatched pairing by displaying G-specific fluorescence enhancement.

Full text of DOI:10.1039/c0ob00413h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Fluorescent nucleoside analogue displays enhanced emission upon pairing with
guanine†
Yun Xie, Tucker Maxson and Yitzhak Tor*
Received 11th July 2010, Accepted 6th September 2010
DOI: 10.1039/c0ob00413h
Table 1 Photophysical data of nucleoside 2a
A fluorescent nucleobase analogue, 7-aminoquinazoline-2,4-
(1H,3H)-dione, is incorporated into a DNA oligonucleotide
and senses mismatched pairing by displaying G-specific
fluorescence enhancement.
d
Solvent
ET(30)b
labs c/nm
l
em/nm
Irel
Water
65.3
55.7
45.9
40.9
36.4
316
316
316
316
316
361
352
339
338
336
1.0
3.2
3.1
2.6
2.9
Methanol
Acetonitrile
Dichloromethane
Dioxane
Single nucleotide polymorphisms (SNPs),1 mutated base pairs,
have been linked to specific diseases or susceptibility to particular
therapeutics.2 While there are several developed and commercial-
ized approaches for detecting SNPs,3 many recent advancements
have centered around the design of base-discriminating fluorescent
nucleosides.4–7 Following incorporation into DNA hybridization
probes and duplex formation with target oligonucleotides, the
emissive nucleosides display characteristic photophysical signa-
ture, depending on their pairing partner.4,8
a Conditions for absorption and emission spectra: 5.0 and 0.5 ¥ 10-5 M,
respectively. b Units are kcal mol-1. c The lowest energy maximum is given.
d Relative emission intensity with respect to intensity in water.
To develop base discriminating probes, it is important to identify
heterocycles that are structurally similar to native nucleobases and
capable of Watson–Crick pairing. Red shifted absorption spectra
relative to native nucleosides, permitting selective excitation,
are highly desirable. The emission of the fluorescent analogs
should be sensitive to its hybridization microenvironment, and
perhaps more importantly, fluorescence enhancement rather than
quenching should be associated with positive identification of
a mismatch. Detecting mismatched G residues has, therefore,
presented a challenge, as guanine, being the easiest nucleobase
to oxidize,9–10 frequently quenches the emission of most com-
monly used fluorophores.11–14 Here we present a new fluorescent
pyrimidine analog that, when hybridized against G, displays an
enhanced emission when compared to a perfect duplex or all other
mismatches.
Scheme
1 Synthesis of the nucleoside and phosphoramidite
based on 7-aminoquinazoline-2,4-(1H,3H)-dione. Reagents: (a)
(i) (NH4)2SO4, N,O-bis(trimethylsilyl)acetamide, CF3SO3Si(CH3)3,
2-D-3,5-di-O-p-toluoyl-a-L-erythro-pentofuranosyl chloride, CH3CN; (ii)
conc. NH4OH, 40%. (b) (i) (CH3)3SiCl, phenoxyacetic anhydride, H2O,
conc. NH4OH, pyridine, 75%; (ii) DMTrCl, Et3N, pyridine, 85%; (iii)
iPr2NEt, (iPr2N)P(Cl)O-CH2CH2CN, ClCH2CH2Cl, 65%.15
In accordance with our design principles,5–7 we have synthesized
a polarizable nucleobase, 7-aminoquinazoline-2,4-(1H,3H)-dione
1 and the corresponding 2¢-deoxynucleoside 2, which contain
an electron-rich ring fused into an electron-deficient pyrimidine
(Scheme 1). We surmise that placing the electron donating amine
group in a conjugated position to the pyrimidine’s carbonyl would
facilitate a charge transfer transition and greater sensitivity of the
photophysical characteristics to environmental changes. To assess
the nucleoside’s sensitivity to its microenvironment, its absorption
and emission spectra were recorded in solvents of distinct polarity
(Fig. 1 and Table 1). Solvent polarity has little effect on the lowest
energy absorption maximum of nucleoside 2 (316 1 nm), but the
Department of Chemistry and Biochemistry, University of California, San
Diego, La Jolla, CA, 92093-0358, USA. E-mail: ytor@ucsd.edu; Fax: +1
858 534 0202; Tel: +1 858 534 6401
† Electronic supplementary information (ESI) available: Details of syn-
thesis, characterization of compounds, thermal melting data and ad-
ditional fluorescence data. CCDC reference number 784395. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c0ob00413h
Fig. 1 Absorption (—) and emission (—) spectra of nucleoside 2 in
water (blue), methanol (green), acetonitrile (red), dioxane (orange), and
dichloromethane (black).
absorption band around 288 nm is sensitive to polarity changes,
resulting in a greater molar absorptivity in nonpolar solvents.
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Table 2 Photophysical data of oligonucleotide 4 and its duplexesa
Importantly, both emission wavelength and intensity are af-
fected by solvent polarity. In water, the most polar solvent
examined, 2 exhibits the most quenched and bathochromically
Duplexes
em/nm
4 · 5
4 · 6
4 · 7
4 · 8
shifted emission band (Fig. 1), peaking around 361 nm (UF
=
l
353
2.2
50
362
1.3
51
361
1.0
57
365
0.8
50
0.039 0.006, Stoke Shift = 3.9 ¥ 103 cm-1). In methanol, nucleoside
2 displays the most intense emission with an emission band at
352 nm (UF = 0.14 0.01, Stoke Shift = 3.2 ¥ 103 cm-1). In
solvents of lower polarity, 2 shows more hyperchromically shifted
emission with decreasing intensity (Table 1, Stoke Shifts = 1.9–
2.1 ¥ 103 cm-1). These observations suggest an enlarged dipole and
charge transfer character of the excited state when compared to
the ground state.
b
Irel
TM ◦C-1
1
1
1
1
a Conditions: 5.0 ¥ 10-6 M in 2.0 ¥ 10-2 M Na3PO4, pH 7.0. b Relative
emission intensity with respect to intensity of 4 · 7.
Table 2). Other oligonucleotides with mismatches (6 and 8) failed
to produce a dramatic increase in fluorescence intensity and all
displayed emission bands around 362 nm, where nucleoside 2
emits in water. Importantly, thermal denaturation measurements
(Table 2 and Figure S4†),15 determined by monitoring changes in
absorbance at 260 nm as a function of temperature, show that
stable duplexes were formed for all oligonucleotide pairs. The
To incorporate the non native nucleoside into a DNA
oligonucleotide, phosphoramidite 3 was prepared (Scheme 1). 7-
Aminoquinazoline-2,4(1H,3H)-dione 1 was glycosylated to pro-
vide the modified nucleoside 2 after saponification of all esters and
isolation of the b-anomer (X-ray Structure: Figure S1 and Table
S1†).15 Protection of the 5¢-hydroxyl as the 4,4¢-dimethoxytrityl
(DMTr) derivative, followed by phosphitylation of the 3¢-hydroxyl,
provided phosphoramidite 3 (Scheme 1). Standard solid-phase
oligonucleotide synthesis was utilized to prepare the 13-mer DNA
construct 4, where probe 2 was placed in the middle of the sequence
(Fig. 2). The oligonucleotide was purified by PAGE, and MALDI-
TOF mass spectrometry confirmed its full length and the presence
of the intact emissive nucleoside 2 (Figure S2†).15
Tm value for the complemented duplex 4 · 7 (Tm = 57
1
◦C)
was within error of the melting temperature of an unmodified
◦
control duplex (Tm = 58 1 C) (Figure S3–S4†). Hybridization
with DNA strands containing mismatches do show, as expected,
destabilization (Table 2).
Nucleoside 2 uniquely reports the presence of a G mismatch
with over a two-fold enhanced emission, compared to its emission
intensity in a perfect duplex when found opposite A, a fea-
ture rarely seen with isosteric/isomorphic fluorescent nucleoside
analogs.11–14 While the underlying molecular factors governing this
behavior are unclear at present, a disparity between the redox
potential of G and the new nucleobase, coupled to environmental
factors influencing the solvation of the modified base are likely to
be influencing factors. It is tempting to speculate that a formation
of a wobble G·2 pair anchors the emissive nucleoside in a partially
exposed geometry, while still preserving a partially stacked and
desolvated microenvironment.16–18 Regardless of these putative
structural features, the results reported here demonstrate that new
emissive nucleobase analogs can display unique photophysical
features and potentially find utility for mismatch detection.
Fig. 2 Synthesized oligonucleotide 4 and oligonucleotides used in
hybridization and fluorescence experiments.
The fluorescent single strand DNA oligonucleotide 4 exhibits a
similar, albeit broader, emission profile to the nucleoside in water
with an emission band around 361 nm. Upon hybridization to
its complement 7, a quenched emission at 363 nm is observed
(Fig. 3 and Table 2). In contrast, when the fluorescently labeled
DNA oligonucleotide 4 is hybridized with 5, an oligonucleotide
with a G mismatch opposite nucleoside 2, its emission is greatly
enhanced and hyperchromically shifted to 353 nm, displaying an
emission more similar to nucleoside 2 in methanol (Fig. 3 and
Acknowledgements
We thank the National Institutes of Health for their generous
support (GM 069773), the National Science Foundation (in-
strumentation grant CHE-0741968), and Nicholas Greco for his
assistance with MALDI experiments.
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The Royal Society of Chemistry 2010
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