A universal nucleoside with strong two-band switchable fluorescence and sensitivity to the environment for investigating DNA interactions

Article abstract of DOI:10.1021/ja3030388

With the aim of developing a new tool to investigate DNA interactions, a nucleoside analogue incorporating a 3-hydroxychromone (3HC) fluorophore as a nucleobase mimic was synthesized and incorporated into oligonucleotide chains. In comparison with existing fluorescent nucleoside analogues, this dye features exceptional environmental sensitivity switching between two well-resolved fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard. Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-selectively monitored.

Full text of DOI:10.1021/ja3030388

Article
pubs.acs.org/JACS
A Universal Nucleoside with Strong Two-Band Switchable
Fluorescence and Sensitivity to the Environment for Investigating
DNA Interactions
Dmytro Dziuba,† Viktoriia Y. Postupalenko,‡ Marie Spadafora,† Andrey S. Klymchenko,‡
Vincent Guerineau,§ Yves Mely,‡ Rachid Benhida,*,† and Alain Burger*,†
́
́
†Institut de Chimie de Nice, UMR 7272, Universite
́
de Nice Sophia Antipolis, CNRS, Parc Valrose, 06108 Nice cedex 2, France
‡Laboratoire de Biophotonique et Pharmacologie, UMR 7213, Faculte
Rhin, 67401 Illkirch, France
́ ́
de Pharmacie, Universite de Strasbourg, CNRS, 74 Route du
§Centre de recherche de Gif, Institut de Chimie des Substances Naturelles CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex,
France
S
* Supporting Information
ABSTRACT: With the aim of developing a new tool to
investigate DNA interactions, a nucleoside analogue incorpo-
rating a 3-hydroxychromone (3HC) fluorophore as a
nucleobase mimic was synthesized and incorporated into
oligonucleotide chains. In comparison with existing fluorescent
nucleoside analogues, this dye features exceptional environ-
mental sensitivity switching between two well-resolved
fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high
fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard.
Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking
bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-
selectively monitored.
fluorophores exhibit two excited states: the initially excited
normal form (N*) and the tautomeric form (T*); each form
generates one well-resolved emission band (Figure 1).4
INTRODUCTION
■
Designing DNA-based fluorescent structures with site-specific
responses to intermolecular interactions is a great challenge.
The majority of environmentally sensitive fluorescent nucleo-
sides are single-band emitters that respond to environmental
changes by changes in the fluorescence intensity or a shift in the
emission maximum.1 These nucleosides suffer from limitations
such as quenching by neighboring nucleobases or poor
sensitivity to perturbations in the DNA structure. For example,
2-aminopurine (2-AP), the most popular nucleotide mimic,1b is
used as an intensiometric probe but exhibits low sensitivity to
the environment and low quantum yields in DNA duplexes.2
These drawbacks have stimulated further research combining
top achievements in photophysics, spectroscopy, and synthetic
chemistry.1b It is particularly important that the spectrum of the
artificial nucleobase be highly sensitive to intermolecular
interactions. Ideally, a single dye should be employed to
avoid double labeling that is needed for fluorescence resonance
energy transfer (FRET), excimers, or J-aggregates.3 In addition,
the effect of the nucleotide mimic on the double-helical
structure of native DNA must be minimal.
Figure 1. ESIPT reaction in 3-hydroxychromones. BPT denotes back
proton transfer, and N* and T* represent the normal and tautomeric
emissive forms, respectively.
The dual emission of 3HCs is highly sensitive to the
environment because an increase in the donating ability of the
hydrogen bond and the dielectric constant of the solvent inhibit
Therefore, we addressed the unique photophysical properties
of 3-hydroxychromone (3HC) dyes, which exhibit polarity- and
hydration-sensitive switching between well-resolved highly
intense fluorescence bands in the visible range. As a result of
excited-state intramolecular proton transfer (ESIPT), these
Received: March 30, 2012
Published: May 16, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Article
Figure 2. Hydrogen-bonding- and dielectric-constant-sensitive nucleoside 1 bearing 2-thienyl-3-hydroxychromone as a base surrogate and its
incorporation into DNA.
Table 1. Thermal Denaturation of Double-Stranded ODN Samples
a
a
a
entry
dsDNA
Tm (°C) ΔTm (°C) entry
dsDNA
Tm (°C) ΔTm (°C) entry
dsDNA
Tm (°C) ΔTm (°C)
1
2
AAA + TTT
AAA + TAT
AAA + TGT
AAA + TCT
AAA + TAbT
AMA + TTT
AMA + TAT
AMA + TGT
AMA + TCT
AMA + TAbT
M-TAT + ATA
50.6
39.2
40.7
39.8
33.7
41.4
41.2
41.4
43.1
41.0
55.5
0
12
13
14
15
16
17
18
19
20
21
TAT + ATA
TAT + AAA
TAT + AGA
TAT + ACA
TAT + AAbA
TMT + ATA
TMT + AAA
TMT + AGA
TMT + ACA
TMT + AAbA
50.8
42.1
42.8
40.9
33.1
45.8
46.3
45.3
46.5
46.6
0
−8.7
−8
−9.9
−17.7
−5
−4.5
−5.5
−4.3
−4.2
22
23
24
25
26
27
28
29
30
31
CAC + GTG
CAC + GAG
CAC + GGG
CAC + GCG
CAC + GAbG
CMC + GTG
CMC + GAG
CMC + GGG
CMC + GCG
CMC + GAbG
55.1
45.8
50.3
47.2
40.5
48.9
49.9
50.0
51.5
51.3
0
−11.4
−9.9
−10.8
−16.9
−9.2
−9.4
−9.2
−7.5
−9.6
+4.7
−9.3
−8.8
−7.9
−14.6
−6.2
−5.2
−5.1
−3.6
−3.8
3
4
5
6
7
8
9
10
11
a
In pH 7 buffer (10 mM cacodylate, 150 mM NaCl).
the ESIPT reaction and thus decrease the relative intensity of
the T* band.5 A critical advantage of ratiometric dyes over
conventional single-band dyes is that this ratio depends only on
the microenvironment of the fluorophore and not on the local
concentration of the dye or the instrument settings. In addition
to the ratio of the two emission bands, information could also
be extracted from the positions of the absorption and two
emission maxima.6 3HCs have been shown to be powerful
fluorescent tools to probe lipid bilayers, cell membranes,
proteins, and peptides,7 but the synthesis of DNA labeled with
ratiometric 3HC derivatives and their application for sensing
have never been described. Our strategy relies on a thienyl-
3HC-modified deoxyribose 1 that allows their incorporation
into oligonucleotides (ODNs) (Figure 2).
We previously described the synthesis and photophysical
properties of such nucleoside analogues.8 Derivative 1 displays
the most promising photophysical properties as a polarity- and
hydration-sensitive label for incorporation into ODNs. The
thienyl-3HC group of 1 is an attractive analogue of nucleic
bases because it is a flat molecule and its size corresponds well
to the size of the two complementary AT and GC base pairs.
We preferred to graft the dye to deoxyribose at the thienyl
substituent rather than to the benzene ring of the chromone.
This grafting should favor the exposure of the 4-carbonyl
moiety to local water in singly labeled ODNs (Figure 2).
Annealing with complementary ODNs or formation of a
protein−DNA complex should reduce the local hydration and
polarity of the dye and thus change the ratio of the intensities of
the two emission bands of 1. Herein we report the synthesis of
ODNs incorporating 1, the photophysical properties of the
labeled ODNs, and the ability of 1 to sense the DNA
microenvironment and DNA−protein interactions site-selec-
tively.
RESULTS AND DISCUSSION
■
The syntheses of the phosphoramidite of 1 and the labeled 15-
and 16-mer ODNs is described in the Supporting Information.
The 15-mer d(CGT TTT XMX TTT TGC) and 16-mer 5′-
d(M CGT TTT TAT TTT TGC) sequences containing
thienylchromone 1 at the positions labeled M were chosen as
model sequences to characterize the photophysical properties
of 1 in ODNs. In the 15-mers (AMA, TMT, CMC and GMG),
1 was incorporated in the middle of the sequence where the
influence of the nucleoside analogue on the thermal stability
should be the most pronounced. Moreover, the sequences
differed by the residues flanking the modified nucleotide to
compare the properties of label 1 in different ODN contexts. In
the 16-mer (M-TAT), the ODN contained a dangling 1 at the
5′ end.9 Control experiments were conducted with reference
wild-type strands of composition d(CGT TTT XAX TTT
TGC), where X = A, C, G or T. Each ODN was then annealed
with its complementary ODN (noted in italics) containing a
natural base or an abasic site located at the position opposite
dye 1, resulting in duplexes with different compositions (see
Table S1 in the Supporting Information). The sequences of the
complementary strands were d(GCA AAA YXY AAA ACG), in
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Journal of the American Chemical Society
Article
which Y is A, C, G, or T and X is any of the natural bases or an
abasic site (Ab).
Table 2. Spectroscopic Data for Nucleoside 1 and Labeled
ODNs
As a first step to characterize the ODNs containing
thienylchromone 1, the thermodynamic stabilities of their
duplexes were compared with the stabilities of the correspond-
ing unlabeled duplexes by monitoring the temperature-induced
absorbance changes at 260 nm (Table 1 and Figure S4 in the
Supporting Information). Thermal denaturation studies
showed that incorporation of nucleoside analogue 1 opposite
a natural base or an abasic site led to duplexes with comparable
thermal stabilities (entries 6−10, 17−21, and 27−31). Although
less stable than the corresponding fully canonical duplexes,
those containing 1 are more stable than the nonlabeled
duplexes with mismatches (Table 1). For example, the duplexes
containing the natural pair (TAT + ATA), the mismatched pair
(TAT + AAA), and dye 1 (TMT + ATA) melted with Tm
values of 50.8, 42.1, and 45.8 °C, respectively (entries 12, 13,
and 17). The data indicate that the dye stacks more strongly
than the natural bases, likely as a result of a larger area of
contact with the surrounding bases. A similar effect was
observed in previous studies with labeled ODNs containing
aromatic groups (e.g., pyrene, biphenyl, bipyridine, isoquino-
lone) instead of the natural base.10 Thus, since thienyl-3HC 1 is
nondiscriminating toward natural bases and less destabilizing
than a mismatch, thienyl-3HC shows characteristics of a
universal base.11 Relative to the canonical duplexes, TMT +
AXA and CMC + GXG gave smaller differences in Tm (ΔTm)
than the AMA + TXT duplexes (Table 1). The ΔTm values can
be grouped in two sets. One is in the range of 3.6−6.2 °C, and
the other has values of 7.5−9.4 °C. The more favorable ΔTm
values for the TMT + AXA and CMC + GXG duplexes might
be due to an increased overlap between the chromone moiety
and the flanking purines of the complementary strand, as
proposed for ODNs incorporating hydroxyphenylbenzoxa-
zole.12 Furthermore, in comparison to the natural base adenine,
1 enhanced the Tm of duplexes containing an abasic site by ∼7
°C for AMA + TAbT and 11−13 °C for TMT + AAbA and
CMC + GAbG (Table 1). The stacking ability of dye 1 was
further confirmed by the 4.7 °C increase in Tm observed for the
M-TAT + ATA duplex containing the dangling thienyl-3HC
(Table 1).9
λ
λN*
λT*
QY
f
a
abs b
c
d
e
entry
sample
(nm)
(nm)
(nm)
I
N*/IT*
(%)
g
1
2
1
367
376
376
376
375
376
377
373
373
376
373
373
373
373
375
381
373
375
375
374
374
372
440
440
441
439
439
438
437
435
437
434
433
433
433
434
434
429
433
434
433
433
438
437
515
541
540
542
542
543
542
540
540
540
541
540
541
543
540
519
540
540
540
541
542
532
1.72
0.11
0.17
0.09
0.10
0.09
0.07
0.23
0.14
0.07
0.09
0.07
0.08
0.28
0.41
0.10
0.43
0.45
0.39
0.66
0.59
1.05
4.6
17
24
20
16
25
30
14
38
28
13
30
35
4
AMA
3
AMA + TTT
AMA + TAT
AMA + TGT
AMA + TCT
AMA + TAbT
TMT
4
5
6
7
8
9
TMT + ATA
TMT + AAA
TMT + AGA
TMT + ACA
TMT + AAbA
CMC
10
11
12
13
14
15
16
17
18
19
20
21
22
CMC + GTG
CMC + GAG
CMC + GGG
CMC + GCG
CMC + GAbG
GMG
1
2
1
1
1
2
M-TAT
8
M-TAT +
ATA
1
a
b
In pH 7 buffer (10 mM cacodylate, 150 mM NaCl). λabs is the
position of the absorption maximum. λN* is the position of the
fluorescence maximum of the N* band. λT* is the position of the
fluorescence maximum of the T* band. IN*/IT* is the ratio of the
intensities of the two emission bands at their maxima. QY is the
fluorescence quantum yield calculated using quinine sulfate (QY =
0.577 in 0.5 M H2SO4) as a reference. Data taken from ref 8, where a
c
d
e
f
g
pH 6.5 buffer (10 mM phosphate) was used.
In a second step, the influence of 1 on the secondary
structure of the labeled duplexes was studied by circular
dichroism (CD) spectroscopy (Figure S5 in the Supporting
Information). The CD spectra were similar to those of the
unlabeled duplexes, indicating that the dye does not affect the
B-helix conformation of the duplexes. Altogether, the thermal
denaturation and CD data support the idea that thienyl-3HC 1
can replace any natural nucleobase or base pair, giving stable
duplexes with the B conformation.
In a third step, we characterized the UV absorbance and
fluorescence properties of 1 in the 15- and 16-mer ODNs,
either in their single-stranded (ss) or double-stranded (ds)
states (Table 2 and Figure 3). In buffer, labeled ssODNs and
dsODNs showed absorption bands centered at 373−376 and
372−381 nm, respectively. The absorption maxima of the
labeled ssODNs and dsODNs were red-shifted by 5−14 nm
relative to that of the free dye in buffer, suggesting stacking of
the dye with its flanking bases (Table 2 and Figure S6 in the
Supporting Information). A red shift of the absorption
maximum is commonly observed with intercalating dyes (e.g.,
acridine and phenanthridinium).13 In ssODNs, the effect of
flanking bases on the 3HC absorption maximum was larger
Figure 3. Normalized fluorescence spectra of (A) free thienyl-3HC 1
in different solvents8 and (B) labeled ssODNs in pH 7 buffer (10 mM
cacodylate, 150 mM NaCl).
with purines than pyrimidines, suggesting a stronger stacking
with purines because of their larger size.
The labeled ODNs showed well-resolved two-band emission,
with the short- and long-wavelength maxima centered at 429−
441 and 519−543 nm, respectively (Table 2). These values are
very close to those obtained for free 1 in various organic
solvents (Figure 3). Thus, the short- and long-wavelength
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Article
bands were assigned to the emissions of the N* and T* forms,
respectively, indicating that the 3HC dye still undergoes an
ESIPT reaction in ODNs and therefore maintains its
fundamental properties. The Stokes shifts for the first and
second emission bands were ∼3870 and ∼8100 cm−1,
respectively. The large Stokes shift obtained for the second
emission band is typical for ESIPT probes.
Interestingly, the fluorescence quantum yield (QY) of the
dye in single- and double-stranded ODNs varied from 1 to 38%
depending on the surrounding nucleobases (Table 2). In
ODNs with A or T flanking the dye, the quantum yields of the
ss and ds forms (entries 2−13) were 3−8-fold larger than that
of the free dye 1 in buffer.
dye 1 show high sensitivity to the neighboring bases, which is a
unique feature with respect to the existing fluorescent bases.1b
Furthermore, the photostabilities of nucleoside 1 and its
ssODN in buffer were comparable to that of prodan in ethanol,
whereas for the corresponding dsODN the photostability was
remarkably higher (Figure S7 in the Supporting Information).
Finally, to validate the application of the new fluorescent base
as a probe for sensing biomolecular interactions, we studied the
interaction of the labeled ODNs with the HIV-1 nucleocapsid
protein (NC). This nucleic acid chaperone protein can locally
destabilize ssODNs or hairpins through interactions with the
hydrophobic amino acids of its folded zinc finger motifs.17
Addition of NC to ssODNs (AMA, TMT, and CMC)
significantly increased their N*/T* intensity ratios, showing a
1:1 interaction stoichiometry (Figure 4 and Figure S8 in the
Supporting Information).
Moreover, with the exception of G opposite 1 (Table 2,
entries 5 and 11), the quantum yields were higher in duplexes
than in single strands and superior to the highest value
determined for free 1 in aprotic solvents (QY = 20% in
dimethyl sulfoxide).8 These observations are consistent with
the dye being shielded in the duplex, with reduced quenching
by the solvent. In addition, reduced flexibility and/or rotation
around the single bond between the chromone and thienyl
moieties might also contribute to the higher quantum yield, as
proposed for thiazole orange, green fluorescent protein (GFP)-
like, and uracil-containing pyridine fluorophores.14 The
quantum yields in ODNs with C or G flanking the dye were
lower (entries 14−22). The decrease in quantum yield in
duplexes with G opposite 1 confirmed the propensity of G to
quench the dye (Table 2, entries 5, 11, and 17). Quenching of
fluorescent nucleoside analogues in ODNs is common.1b For
example, G, T, and C efficiently quench pyrene,15 while 2-AP is
quenched by all four natural nucleobases.2 Importantly, the
fluorescence quantum yield of 1 is 2−25-fold larger than that of
2-AP in corresponding ODN sequences.16 In addition, the
molar absorptivity of 1 (13 000 M−1 cm−1) is about twice that
of 2-AP (7200 M−1 cm−1), which makes it up to 50-fold
brighter.8,16
In comparison to free dye 1 in buffer, the labeled ODNs
showed a strong decrease in the N*/T* intensity ratio (IN*/
IT*) together with blue and red shifts of the N* and T* bands,
respectively (Table 2 and Figure 3). For most of the labeled
ODNs, IN*/IT* was between those for the free dye in
dichloromethane and acetonitrile (Figure 3), indicating that
the environment of the labeling site is mainly aprotic and of
medium polarity.8 Here we compared only ODNs showing
sufficient fluorescence quantum yields (≥4%; entries 2−14). In
ssODNs, IN*/IT* varied strongly with the nature of the
neighboring bases. TMT and CMC showed larger IN*/IT*
than AMA, likely as a consequence of more efficient stacking
of dye 1 with purines and thus more efficient shielding from
water. These results are consistent with the UV spectroscopy
data, where the more pronounced red shift of the 3HC
absorption maximum was observed with flanking adenines in
AMA (see above). In dsODNs, IN*/IT* rather uniformly
showed lower values, with the exception of T opposite 1 in
both the AMA and TMT duplexes (entries 3 and 9). It is clear
that in dsDNA, the environment of 1 is highly dehydrated,
which explains the low IN*/IT* values. The anomalous effect of
having T opposite 1 could be explained by H-bonding between
this base and the 4-carbonyl of 1, which would hamper the
ESIPT and increase IN*/IT*. Finally the highest N*/T*
intensity ratio was observed for M-TAT ssDNA, where dye
1, located at the 5′ end, is more exposed to water. Thus, both
the fluorescence quantum yield and IN*/IT* for thienyl-3HC
Figure 4. Fluorescence spectra of the TMT ODN in the absence (red)
or presence of 1 equiv (blue solid) or 3 equiv (blue dotted) of NC or 3
equiv of NC-SSHS (black dashed) peptides. The spectra were
normalized at the T* band. The experiments were performed in pH 7
buffer (10 mM cacodylate, 150 mM NaCl). Binding of the NC peptide
to the labeled ODN leads to an increase in IN*/IT* due to the insertion
of the hydrophobic amino acids of its folded finger motifs between the
bases.
As already described for several other NC−ODN complexes,
these observations suggest that intercalation of the zinc finger
residues between the bases of these labeled ssODNs, at the
level of 1, decreases the base stacking and increases the
exposure of the bases to water.18 In contrast, no change in the
probe signal was observed upon interaction of NC with the
labeled dsODNs (data not shown) because of the limited
destabilizing effect of NC on stable ds regions.19 Moreover, a
nonfolded mutant of NC (NC-SSHS) that binds ssODNs only
through electrostatic interactions with the ODN phosphate
groups18a,20 induced no change in the emission spectra of the
labeled ODN sequences (Figure 4). These data confirmed that,
in contrast to NC, the NC-SSHS mutant does not affect the
base stacking in ssODNs. Thus, our new fluorescent base allows
site-selective monitoring of subtle conformational changes
produced by interacting proteins.
CONCLUSION
■
ODNs incorporating the two-band fluorescent thienyl-3HC 1
were successfully synthesized. Dye 1 can replace any natural
nucleobase or base pair to form duplexes with the B
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Journal of the American Chemical Society
Article
(7) (a) Demchenko, A. P.; Mely, Y.; Duportail, G.; Klymchenko, A. S.
Biophys. J. 2009, 96, 3461−3470. (b) Shynkar, V. V.; Klymchenko, A.
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conformation having nearly the same stability as the native
nonlabeled duplex. Thus, 1 possesses the characteristics of a
universal base. In comparison to the “gold standard” 2-AP, the
new base is up to 50-fold brighter, and its absorption is red-
shifted by 60 nm. Consequently, the absorption of the new base
does not overlap with the intrinsic absorptions of nucleic acids
and proteins. The new fluorescent base demonstrates all the
advantages of wavelength ratiometric detection, including a
large separation between the emissions of the two bands and a
dramatic variation of their relative contributions. Moreover, the
high sensitivity of the dual emission of 1 to the polarity of the
environment induces a strong shielding effect of the flanking
bases from water. Finally, the new base was successfully applied
to monitor local conformational changes of ssODNs upon
interaction with the viral nucleocapsid protein. Therefore, the
new base appears to be a new universal tool for DNA research.
Further chemical modifications of 3HC nucleobases will
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ASSOCIATED CONTENT
* Supporting Information
■
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F.; Thuong,
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material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
rachid.benhida@unice.fr; burger@unice.fr
Weinhold, E.; Seitz, O. Pure Appl. Chem. 2005, 77, 327−338.
(b) Wenge, U.; Wagenknecht, H.-A. Synthesis 2011, 502−508.
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■
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the ANR (07-BLAN-0287) and the FRM
(DCM20111223038) for financial support and grants for
D.D. and V.Y.P. We thank Prof. Alexander Demchenko and Dr.
Jennifer Wytko for their fruitful comments and critical reading
of the manuscript.
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