Highly fluorescent guanosine mimics for folding and energy transfer studies

Article abstract of DOI:10.1093/nar/gkr281

Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photop

Full text of DOI:10.1093/nar/gkr281

Published online 6 May 2011
Nucleic Acids Research, 2011, Vol. 39, No. 15 6825–6834
doi:10.1093/nar/gkr281
Highly fluorescent guanosine mimics for folding
and energy transfer studies
¨
Anaelle Dumas and Nathan W. Luedtke*
Institute of Organic Chemistry, University of Zu¨ rich, Winterthurerstrasse 190, CH-8057 Zu¨ rich, Switzerland
Received February 20, 2011; Revised April 11, 2011; Accepted April 12, 2011
INTRODUCTION
ABSTRACT
From the early days of structural biology, DNA was
recognized as having the ability to adopt alternate folds
(1). In addition to A-, B- and Z-form double helices, DNA
can fold into a variety of hairpin, triplex, G-quadruplex
and i-motif structures that contain non-canonical base
pairs (2,3). While the exact biological relevance of these
structures remains an open question, DNA sequences with
the ability to fold into G-quadruplex structures have been
implicated in regulating gene transcription (4–6), recom-
bination (7), chromosome stability (8–10) and
programmed cell death (11). In all cases, open questions
still remain about the exact folding pathways and
biologically active conformation(s) of these DNA
sequences (12).
A wide variety of biophysical techniques have been used
to characterize DNA folding in vitro. These include NMR
(13–15), circular dichroism (CD) (16), UV absorption
(17,18), X-ray crystallography (19), FRET (20–22) and
immunostaining (23). Most of these methods require
pure DNA samples and are not compatible with conform-
ational analyses in living cells. While fluorescence spec-
troscopy has the potential for conformational analyses
in vivo, techniques involving high-affinity fluorescent
probes or DNA molecules conjugated to large fluorescent
tags can perturb the structure and/or stability of the DNA
itself (21,24,25). Further development of fluorescence-
based methods is needed to provide highly sensitive,
non-perturbing tools to characterize DNA folding
in vitro and in vivo. Due to their small size and predictable
location, internal fluorescent probes offer some advan-
tages over linker-attached chromophores, such as the
ability to report subtle structural changes with single nu-
cleotide resolution (26–29).
Guanosines with substituents at the 8-position can
provide useful fluorescent probes that effectively
mimic guanine residues even in highly demand-
ing model systems such as polymorphic
G-quadruplexes and duplex DNA. Here, we
report the synthesis and photophysical properties
of
a
small family of 8-substituted-20-deoxy-
guanosines that have been incorporated into
the human telomeric repeat sequence using
phosphoramidite
chemistry.
These
(2PyG),
include
8-(2-
8-(2-pyridyl)-20-deoxyguanosine
phenylethenyl)-20-deoxyguanosine (StG) and 8-[2-
(pyrid-4-yl)-ethenyl]-20-deoxyguanosine (4PVG). On
DNA folding and stability, 8-substituted guanosines
can exhibit context-dependent effects but were
better tolerated by G-quadruplex and duplex struc-
tures than pyrimidine mismatches. In contrast to
previously reported fluorescent guanine analogs,
8-substituted guanosines exhibit similar or even
higher quantum yields upon their incorporation
into nucleic acids (( = 0.02–0.45). We have used
these highly emissive probes to quantify energy
transfer efficiencies from unmodified DNA
nucleobases to 8-substituted guanosines. The
resulting DNA-to-probe energy transfer efficiencies
(gt)
are
highly
structure
selective,
with
gt(duplex) < gt(single-strand)
<
gt(G-quadruplex).
These trends were independent of the exact
structural features and thermal stabilities of the
G-quadruplexes or duplexes containing them.
The combination of efficient energy transfer, high
probe quantum yield, and high molar extinc-
tion coefficient of the DNA provides a highly
sensitive and reliable readout of G-quadruplex
formation even in highly diluted sample solutions
of 0.25 nM.
Modification of the 8-position of guanosine is an at-
tractive avenue to fluorescent derivatives because it is
not directly involved in base-pairing interactions within
G-quadruplex or duplex structures (R2, Figure 1A).
Derivatization of this position can generate fluorescent
products (Figure 1B) (30–34) with emission properties
*To whom correspondence should be addressed. Tel: +41 44 635 4244; Fax: +41 44 635 6891; Email: luedtke@oci.uzh.ch
ß The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

6826 Nucleic Acids Research, 2011, Vol. 39, No. 15
H
R
1
A
B
N
N
R
1
H
O
N
N
N
R
2
λ
abs
/
λ
em
(nm) Φ (CH CN) Φ (H O)
Φ (D O)
Entry
3
2
2
N
H
N
N
-4
H
O
"dG" (1)
N
260 / 330
300 / 415
n.d
< 10
0.02
n.d
H
H
H
N
N
N
H
+
H
O
6
N
H
N
7
O
N
1
N
H
0.71
0.74
0.57
0.04
"2PyG" (2)
"StG" (3)
"4PVG" (4)
8
N
R
2
4
2
N
N
1
H
R
1
N
N
H
R
340 / 450
355 / 490
0.49
0.16
0.50
0.30
Guanosine / G-tetrad
R = 2'-deoxyribonucleoside (1 - 4)
N
1
R = DNA oligonucleotide (hTelo)
1
Figure 1. (A) Structures and numbering of 8-substituted-20-deoxyguanosines and an ion-containing G-tetrad (gray). (B) Wavelengths of maximal
absorbance (labs) and emission (lem) in water, and quantum yield (È) of each nucleoside in acetonitrile, water or D2O.
that are sensitive to DNA folding (34). While the addition
of bulky groups to the 8-position of guanosine can shift
the conformational equilibrium of the glycosidic bond
from ‘anti’ to ‘syn’ (35–38), DNA folding can force
8-modified guanosines to adopt anti conformations with
relatively small energetic penalties to DNA folding
(ÁÁG ꢀ 1 kcal molꢁ1) (37,38).
of cation-containing G-quadruplexes are therefore respon-
sible for the efficient energy transfer reactions within these
structures (34). These phenomena together provide a
reliable and highly sensitive means for deciphering the
folded state of the DNA (duplex versus quadruplex)
even in highly diluted sample solutions of ꢀ250 pM.
We recently reported 8-(2-pyridyl)-20-deoxyguanosine
‘2PyG’ (Entry 2, Figure 1B) as a ‘turn-on’ fluorescence
probe of G-quadruplex folding and energy transfer (34).
Upon its incorporation into G-tetrads, 2PyG was minim-
ally disruptive to intramolecular G-quadruplex folding
where it exhibited higher quantum yields (È = 0.04–
0.15) than the corresponding nucleoside (2) in water
(È = 0.02). Using 2PyG, we discovered efficient energy
transfer reactions involving unmodified guanosines in
G-quadruplex structures (DNA-to-probe energy transfer
efficiency, ꢀt = 0.11–0.41) that depended on the presence
and identities of cations coordinated to the O6 positions of
the guanine residues. It was unclear from these studies,
however, if the reported intramolecular energy transfer
reactions resulted from unusual intrinsic properties of
G-quadruplexes, the unusual photophysical characteristics
of 2PyG, or some combination thereof. To help address
this question, we have synthesized oligonucleotides con-
taining 8-(2-phenylethenyl)-20-deoxyguanosine (StG) and
MATERIALS AND METHODS
Synthesis of 8-substituted-20-deoxyguanosine nucleosides
and phosphoramidites
The 2PyG nucleoside (2) and corresponding b-cyanoethyl
phosphoramidite were synthesized according to pub-
lished procedures (34). The phosphoramidites of
8-(2-phenylethenyl)-
and
8-[2-(pyrid-4-yl)-ethenyl]-
20-deoxyguanosine were synthesized according to
Scheme 1. The 8-bromo-O6-protected-20-deoxyguanosine
derivative 6 is a highly versatile intermediate for palladium
catalyzed cross-coupling reactions including Suzuki, Stille
and Heck. The 8-(2-phenylethenyl)-derivative 7 was
synthesized by Suzuki–Miyaura cross-coupling of 6 with
commercially available trans-styryl boronic acid pinacol
ester to yield the trans isomer of 7. The analogous
pyridine derivative 9, was made by first preparing the
vinyl derivative
8 via Stille coupling of 6 with
8-[2-(pyrid-4-yl)-ethenyl]-20-deoxyguanosine
(4PVG,
tributyl(vinyl) tin, followed by addition of the pyridine
group via Heck coupling. The resulting trans derivatives
7 and 9 were deprotected with tetrabutylammonium
fluoride (TBAF) to furnish free nucleosides (3 and 4,
Figure 1B), or they were carried forward into
phosphoramidite synthesis using standard methodologies
for N(2) protection with isobutyryl chloride (10 and 11),
followed by deprotection with TBAF (12 and 13) and the
addition of dimethoxytrityl to the 50OH groups (14 and
15). The detailed synthetic procedures and characteriza-
tion of these intermediates are available in the supporting
information. Slightly different procedures were needed for
introducing the cyanoethyl phosphoramidite on the 30OH
of each compound (16 and 17). In the case of 17, a base
Figure 1B) and evaluated their ability to report DNA
folding and internal energy transfer reactions in
G-quadruplex, single-stranded and duplex structures
derived from the human telomeric repeat sequence. As
compared to 2PyG, both of the vinyl-bridged derivatives
4PVG and StG exhibit red-shifted excitation and emission
maxima, lower potentials for perturbing DNA structure,
and higher quantum yields (È = 0.03–0.44) in the context
of folded DNA. For all three 8-substituted guanosines,
DNA-to-probe energy transfer efficiencies were much
higher in G-quadruplex structures (ꢀt = 0.12–0.30) than
the same oligonucleotides folded into B-form duplexes
(ꢀt = 0.02–0.06). The unusual photophysical properties

Nucleic Acids Research, 2011, Vol. 39, No. 15 6827
Si
Si
Si
O
O
O
O
O
N
B
Isobutyryl chloride
N
N
N
N
N
N
N
O
Pyridine
r.t., 6h
N
Br
N
N
NH2
N
N
H
Pd(PPh )
NH2
O
O
3 4
O
72%
Na CO 3M
2
3
TBDMSO
TBDMSO
TBDMSO
TBDMSO
(6)
DME, Toluene
80°C, 20h
68%
(7)
(10)
TBDMSO
TBDMSO
Toluene
100°C, 2h
90%
SnBu3
Pd(PPh )
3 4
Si
O
Si
Si
O
N
O
N
Isobutyryl chloride
Pyridine
N
N
O
N
N
N
N
N
N
r.t., 4h
N
Br
N
HCl
N
N
N
H
O
87%
NH2
N
NH2
O
O
Pd(OAc) / (oTol) P
TBDMSO
2
3
(11)
Et N, DMF
100°C, 20h
72%
(9)
3
TBDMSO
(8)
TBDMSO
TBDMSO
TBDMSO
TBDMSO
O
N
R
N
O
N
O
NH
O
(10)
-or-
(11)
2-cyanoethyl diisopropyl
chlorophosphoramidite
R
N
N
R
N
NH
O
N
NH
O
N
H
O
N
DMT
NC
O
N
H
N
N
H
DMT-Cl
-or-
O
O
2-cyanoethyl tetraisopropyl
phosphoramidite
5-ethylthio-1H-tetrazole
DMT
O
TBAF
THF
r.t., 17h
Pyridine
r.t., 1-3h
HO
O
P
HO
HO
O
N(iPr)2
(14) R = Phenyl; 71%
(15) R = 4-Pyridyl; 68%
(16) R = Phenyl; 99%
(17) R = 4-Pyridyl; 82%
(12) R = Phenyl; 95%
(13) R = 4-Pyridyl; 67%
Scheme 1. Synthesis of the 8-(substituted)-20-deoxyguanosine phosphoramidites 16 and 17.
catalyzed reaction with 2-cyanoethyl diisopropylchloro
phosphoramidite was successful, while compound 16 was
prepared by an acid catalyzed reaction of 14 with
2-cyanoethyl tetraisopropyl phosphordiamidite by
5-ethylthio-(1 H)-tetrazole. All new compounds were
characterized by NMR and mass spectrometry
(Supplementary Data), except for compound 16 that was
carried directly into oligonucleotide synthesis without
isolation (39).
prepared in pure water and quantified using e(260
nm) = 244 300 cmꢁ1Mꢁ1
MALDI-TOF MS hTeloG9:
.
G9 = StG (m/z) calcd, 7676.4; found 7684.5;
G9 = 4PVG (m/z) calcd, 7677.4; found 7680.4;
hTeloG23: G23 = StG (m/z) calcd, 7676.4; found 7678.4;
G23 = 4PVG (m/z) calcd, 7677.4; found 7677.8. The syn-
thesis and characterization of the 2PyG-containing oligo-
nucleotides were reported elsewhere (34). See
Supplementary Figures S1–S6 for analytical HPLC chro-
matograms and MALDI-MS spectra.
Oligonucleotide synthesis
DNA folding and buffer conditions
Modified oligonucleotides were synthesized on a 1-mmol
scale using a Millipore Expedite 8909 DNA synthesizer
according to the standard Trityl-off procedure, except
that 5% dichloroacetic acid in CH2Cl2 was used for
Trityl deprotection. Manual coupling reactions were
used for the site-specific introduction of 16 and 17 into
oligonucleotides. These phosphoramidites (0.02 mmoles)
were dissolved into 200 ml of 0.25 M 5-ethylthio-(1 H)-
tetrazole in dry acetonitrile and added to DNA columns
via syringe for 10 min. Upon completion of each sequence,
oligonucleotides were cleaved from the solid support and
deprotected by treatment with 1.5 ml of 33% aqueous
ammonium hydroxide at 55ꢂC for 17 h in a 1.7-ml
screw-top cap tube. The resulting products were dried,
dissolved into water and purified by using C-18 reversed
phase HPLC with a mobile phase of 0.1 M triethyl
ammonium acetate (pH 7) and acetonitrile. Following
lyophilization, oligonucleotide stock solutions were
Duplex DNA samples were prepared in the presence of
100 mM NaCl and 1.1 equivalents of the complementary
strand. G-quadruplex structures were prepared from
single-stranded oligonucleotides in 100 mM of NaCl or
KCl-containing buffers. Unfolded, single-stranded
DNAs were prepared in 100 mM LiCl. Buffers were
prepared as 10 mM cacodylic acid solutions containing
the indicated salt, and the pH was adjusted to 7.4 by neu-
tralization with KOH, NaOH or LiOH. The final buffers
had the following compositions: (K+): 10 mM potassium
cacodylate buffer + 100 mM KCl; (Na+): 10 mM sodium
cacodylate buffer + 100 mM NaCl; (Li+): 10 mM lithium
cacodylate buffer + 100 mM LiCl; duplex sample (DS):
10 mM sodium cacodylate buffer + 100 mM NaCl.
Oligonucleotides were diluted into buffer to final concen-
tration of 2 mM and heated for 5 min at 95ꢂC, and slowly
cooled to room temperature overnight before use.

6828 Nucleic Acids Research, 2011, Vol. 39, No. 15
CD and thermal denaturation studies
quartz cuvette. Instrument settings were maintained for all
reported fluorescence measurements, allowing for direct
comparisons of fluorescence intensities (Figure 2). For
measurements of nucleoside quantum yields (Figure 1B),
optical densities were adjusted to 0.1 0.01 at the
indicated lex of each nucleoside and corresponding
standard. As the standard for 2PyG (lex = 300 nm),
2-aminopyridine in 0.1 N H2SO4 (È = 0.6) was used,
and quinine sulfate in 0.1 M H2SO4 (È = 0.56) was used
as a reference for StG (lex = 340 nm) and 4PVG
(lex = 355 nm). Quantum yields of each probe in the
context of oligonucleotides were determined as above
except that the corresponding nucleosides in water were
used as references. The excitation spectra of each probe in
the context of DNA were collected using the following
wavelengths of emission: 415 nm for 2PyG, 450 nm for
StG and 475 nm for 4PVG. For all measurements
involving oligonucleotides, 2 mM solutions of pre-folded
DNA were used. This allowed for direct comparisons of
CD and fluorescence spectra, but the relatively high ab-
sorbance of DNA samples at 260 nm (ꢃ0.4 AU) caused a
CD spectra were collected on a Jasco model J-715
spectropolarimeter equipped with a Julabo FS 18 tem-
perature control system and a 1.0-cm path length
thermo-controlled CD quartz cell. Spectra were collected
at 25ꢂC between 220 and 400 nm with 0.1 nm steps, 2 nm
band width and scanning rate of 50 nm minꢁ1. Three scans
were averaged for each reported spectrum. For
DNA-melting experiments, 2 mM solutions of pre-folded
DNA were equilibrated for 15 min at 10ꢂC and slowly
ramped to 95ꢂC at a rate of 30ꢂC hꢁ1. The ellipticity of
each sample was monitored at 290 nm for the hTelo
derived G-quadruplexes and 260 nm for the corresponding
duplexes. Reverse temperature scans showed no hysteresis.
Photophysical measurements
Energy transfer efficiencies and quantum yields were
calculated as previously reported (34,40,41). Absorption
and emission spectra were recorded using a Molecular
Devices SpectraMax M5 instrument in a 1-cm path-length
A
B
C
D
E
F
G
H
I
Figure 2. Excitation spectra of 2 mM of StG (A), 4PVG (B) or 2PyG (C) in the presence of 100 mM KCl (K+), 100 mM NaCl (Na+) or 100 mM LiCl
(Li+). Excitation spectra of hTeloG23(StG) (D), hTeloG23(4PVG) (E) and hTeloG23(2PyG) (F) were collected using emission at 450, 475 and
415 nm, respectively. Emission spectra of hTeloG23(StG) (G), hTeloG23(4PVG) (H) and hTeloG23(2PyG) (I) were collected using excitation at
260 nm. Double-stranded samples ‘DS’ included 1.1 equivalents of the complementary strand and were prepared in 100 mM NaCl. All samples
contained 2 mM of DNA or 8-substituted-20-deoxyguanosine nucleoside (2–4) in an aqueous 10 mM cacodylate buffer (pH 7.4).

Nucleic Acids Research, 2011, Vol. 39, No. 15 6829
Table 1. Quantum yield (È), DNA-to-probe energy transfer efficiency (ꢀt) and the ratio of fluorescence intensities obtained upon excitation at
260 nm [F(260)] and at 360 nm [F(360)] for StG, 375 nm [F(375)] for 4PVG and 325 nm [F(325)] for 2PyG
Name
Cond.
StG
4PVG
2PyG
È
ꢀt
F(260)/F(360)
È
ꢀt
F(260)/F(375)
È
ꢀt
F(260)/F(325)
hTeloG9
hTeloG23
Li+
K+
Na+
DS
0.45
0.44
0.37
0.32
0.35
0.33
0.37
0.20
0.06
0.21
0.18
0.02
0.03
0.12
0.12
0.02
2.0
3.5
3.9
0.9
1.9
3.3
3.3
1.3
0.05
0.04
0.03
0.03
0.07
0.04
0.06
0.02
0.17
0.28
0.36
0.04
0.16
0.24
0.31
0.05
2.0
3.3
3.7
1.0
1.8
2.9
3.0
1.2
0.04
0.09
0.08
0.03
0.05
0.05
0.10
0.04
0.09
0.24
0.30
0.03
0.11
0.19
0.26
0.06
2.3
4.0
4.7
1.1
1.8
3.0
3.3
1.4
Li+
K+
Na+
DS
Fluorescence spectra of these same samples are shown in Figure 2. Cond.: Measurements were performed in the presence of 100 mM KCl (K+),
100 mM NaCl (Na+) or 100 mM LiCl (Li+) in an aqueous 10 mM cacodylate buffer (pH 7.4). Double-stranded samples (DS) included 1.1 equivalents
of the complementary strand and 100 mM NaCl.
significant attenuation of the excitation beam that reduced
the fluorescence intensities from each probe. This was
evidenced by non-linear relationships between raw fluor-
escence intensities Fraw(lex) and DNA concentrations
above 50 nM when samples were photoexcited at 260 nm
(Supplementary Figure S7A). Fraw(lex) values at each ex-
citation wavelength lex were therefore corrected by multi-
plication with the following correction factor (CF) to
obtain corrected F(lex) values (40). Where A(lex) is the
absorbance of the sample at each wavelength of
excitation.
photoisomerization and reached a E:Z ratio of 80:20.
According to HPLC and NMR analysis, we were able to
reproduce these results with nucleoside 3 in our laboratory
(42). Replacing the phenyl ring of 3 with a pyridine group
generates a new fluorescent nucleoside derivative trans-8-
(4-pyridyl-vinyl)-20-deoxyguanosine (4) that exhibits little,
if any photoswitching (45). This feature allows simplified
interpretation of CD and fluorescence data collected using
compound 4 in the context of oligonucleotides.
Syn–anti conformational analyses of nucleosides 2–4
Introducing bulky substituents to the C8 position of a
purine residue is known to shift the conformational pref-
erence of the N9-C10 glycosidic bond towards syn (36–38).
2:303 ꢄ AðꢁexÞ
CF ¼
:
ð1Þ
exÞ
1 ꢁ 10ꢁAðꢁ
1
This can be evaluated using H and 13C NMR. A con-
All fluorescence spectra (Figure 2) and ‘F(260)/F(X)’
ratios (Table 1) were measured using 2-mM solutions of
DNA and corrected according to Equation (1). To check
the validity of this approach, serial dilutions of
StG-containing duplex and G-quadruplex DNAs were
analyzed for fluorescence intensity as a function of
sample concentration (Supplementary Figure S7). When
the samples were excited at 260 nm, linear relationships
were observed only after the application of the CF in
Equation (1) (Supplementary Figure S7A–B). At DNA
concentrations of 50 nM and below, the fluorescence
intensities and F(260)/F(X) ratios for the CF-corrected
and uncorrected fluorescence measurements converged to
the same values due to the low absorbance of the samples.
These results validate the use of Equation (1). It should be
emphasized that this CF is not needed for dilute DNA
samples (<50 nM) having minimal optical densities
(OD ꢀ 0.01) at 260 nm.
formational change from anti to syn correlates with a
downfield shift of H20, C10, C30 and C40 and an upfield
shift of the C20 signal (46,47). According to this analysis,
the 2PyG nucleoside 2 prefers a syn conformation. In
contrast, the C8 vinyl bridged compounds 3 and 4
exhibit chemical shifts consistent with an anti conform-
ational preference of the glycosidic bond (Supplementary
Table S1). These preferences were confirmed by
2D-ROESY experiments, where strong cross peaks were
observed between the phenyl/pyridyl rings and 50OH, H50,
H30 and H20 of compounds 3 and 4, but were absent in
2PyG (Supplementary Figures S8–S10). These results
confirm a syn conformational preference for 2PyG, and
anti preference for StG and 4PVG. These conformational
preferences might be important in the context of oligo-
nucleotides, where StG and 4PVG are generally less dis-
ruptive to DNA folding than 2PyG.
Global structure and thermal stabilities of oligonucleotides
RESULTS AND DISCUSSION
In K+-containing solutions, the human telomeric repeat
DNA sequence [G3(T2AG3)3] ‘hTelo’ can fold into
several distinct G-quadruplex conformations in popula-
tions that are largely determined by the 50- and
30-flanking nucleotides (3,48–50). The sequence studied
Cis–trans conformational analyses of nucleosides 3–4
Derivatives of 8-vinyl guanosine, including 8-
(2-phenylethenyl)-20-deoxyguanosine (3), were recently
reported to be cis–trans photoisomerizable switches
capable of influencing DNA conformation (39,42–44)
here (TT[G3(T2AG3)3]A) mostly adopts
a
‘(3+1)
Nucleoside
3
photoisomerizes upon irradiation at
parallel–anti-parallel hybrid’ topology in the presence of
K+ (49). In Na+-containing solutions this same sequence
folds into a ‘anti-parallel’ topology in solution (50,51).
370 nm to reach a photostationary E:Z ratio of 6:94,
while irradiation at 254 nm caused the reverse

6830 Nucleic Acids Research, 2011, Vol. 39, No. 15
A
B
C
D
E
F
Figure 3. CD spectra of single-stranded hTeloG9 and hTeloG23 derivatives in 100 mM LiCl (A), 100 mM KCl (B and C) or 100 mM NaCl (D).
Double-stranded DNA samples ‘DS’ included 1.1 equivalents of the complementary strand and were prepared in 100 mM NaCl (E and F).
All samples contained 2 mM of DNA in an aqueous 10 mM cacodylate buffer (pH 7.4).
Each of these structures contain a characteristic mixture of
syn and anti glycosidic conformations of guanosines
involved in G-tetrad formation (3).
The substitution of G23 with 2PyG caused hTelo to fold
into an ‘anti-parallel’ topology even in K+ solutions
(Figure 3C). This structure, where G23 adopts a syn con-
formation, is normally only observed in Na+-containing
solutions (50,51). hTeloG23(2PyG) was therefore
characterized in Na+ buffer, where it exhibited nearly
the same global structure and thermal stability as the
wild-type sequence in Na+ (ÁTm = ꢁ2ꢂC, Figure 3D
and Table 2). StG and 4PVG, in contrast, did not force
hTeloG23 to fold into an ‘anti-parallel’ structure in K+
solutions, and, like the wild-type sequence, two maxima
in the region between 270 and 305 nm, and a minimum at
ꢃ240 nm were observed (Figure 3C). StG and 4PVG were
also compatible with the formation of the ‘anti-parallel’
hTelo structure in Na+ solutions (Figure 3D). Taken
together, these results suggest that single-stranded hTelo
oligonucleotides containing StG and 4PVG exhibit folding
behavior more similar to wild-type hTelo than 2PyG or
T-containing variants.
In contrast to the mutation-sensitive folding of
single-stranded hTelo, the substitution of position G9 or
G23 with 2PyG, StG, 4PVG or T resulted in little impact
on the global structure of the corresponding duplex DNAs
(Figure 3E and F). Due to the close proximity of G23 to
the end of the oligonucleotide, these modifications had
little impact on thermal stability of the duplex (Table 2).
Duplexes containing modifications at the central G9
position, in contrast, exhibited thermal stabilities that
were highly sensitive to mutations. T incorporation at
G9 caused a ÁTm = ꢁ8ꢂC as compared to the wild type
hTelo duplex (Table 2). The 8-substituted guanosines
caused much less loss in thermal stability than T, with
Folding of single-stranded hTelo oligonucleotides was
controlled by variation of alkali metal salts in the buffer.
The global structures and stabilities of the resulting
mixtures were assessed using temperature-dependent CD.
In Li+-containing solutions, CD data were consistent with
mostly unfolded, G-stacked single-stranded DNA
(Figure 3A). Similar results were obtained for all modifi-
cations introduced at positions G9 and G23 of hTelo in
Li+ buffer. In buffers containing 100 mM KCl, hTelo
adopts a ‘(3+1) hybrid’ structure that exhibits a positive
CD peak at 288 nm with a shoulder at 270 nm and a
minimum at 242 nm (49). The same characteristic CD
spectrum was observed when G9 was replaced with
2PyG, StG and 4PVG, suggesting the presence of
wild-type folds (Figure 3B). In K+ buffers, 2PyG caused
a larger thermal stabilization of the ‘(3+1) hybrid’ struc-
ture (10ꢂC) as compared to StG and 4PVG (5ꢂC and 8ꢂC,
Table 2). In contrast, replacing G9 with T caused very
large changes to the global structure of hTelo in K+
(Figure 3B) that were accompanied by a dramatic
decrease in thermal stability (ÁTm = ꢁ23ꢂC, Table 2).
We interpret these spectral changes and decreased stability
as the loss of one G-tetrad and formation of a
G-quadruplex containing only two G-tetrads with
‘head-to-head’ heteropolar stacking orientation (52).
These results suggest that position G9 is sensitive to
mutation, and that 2PyG, StG and 4PVG are
incorporated directly into the G-tetrads of natively
folded hTelo in the presence of K+.

Nucleic Acids Research, 2011, Vol. 39, No. 15 6831
Table 2. Thermal denaturation melting temperatures (Tm) of
single-stranded oligonucleotides in K+ or Na+, and duplex samples
(DSs) in Na+ as determined by temperature-dependent CD
decreases in quantum yield upon their incorporation
into duplex DNA (55–57). In contrast, 8-substituted
guanosines exhibit relatively little, if any quenching
upon
their
incorporation
into
G-quadruplex,
Name
X
Tm in K+
(ÁTm)
Tm in Na+
(ÁTm)
Tm of DS
(ÁTm)
single-stranded or duplex DNA. The quantum yield of
2PyG in water (0.02) increases to È = 0.03ꢁ0.05 upon
its incorporation into single-stranded or duplex hTelo
DNA, and it increases to È = 0.05ꢁ0.15 when the same
oligonucleotides were folded into G-quadruplex structures
(34). In the case of 4PVG (È = 0.16), somewhat lower
quantum yields were obtained upon its incorporation
into single-stranded
(Èf = 0.03ꢁ0.07) and still lower quantum yields were
observed in the context of duplex DNA
hTelowt
hTeloG9
G
67.7
53.5
66.0
StG
4PVG
2PyG
T
StG
4PVG
2PyG
T
73.1 (5.4)
75.9 (8.2)
77.5 (9.8)
44.6 (ꢁ23)
59.5 (ꢁ8.2)
61.5 (ꢁ6.2)
59.3 (ꢁ8.4)
45.2 (ꢁ23)
60.6 (7.1)
61.1 (7.6)
61.8 (8.3)
39.2 (ꢁ14)
52.7 (ꢁ0.8)
47.5 (ꢁ6.0)
51.6 (ꢁ1.9)
45.1 (ꢁ8.4)
63.0 (ꢁ3.0)
64.6 (ꢁ1.4)
61.2 (ꢁ4.8)
57.8 (ꢁ8.2)
66.5 (0.5)
66.9 (0.9)
66.2 (0.2)
64.5 (ꢁ1.5)
hTeloG23
and quadruplex
structures
CD spectra of these same samples are shown in Figure 3.
ÁTm = Tm(modified oligo) ꢁ Tm(wild type).
(Èf = 0.02ꢁ0.03). Upon its incorporation into DNA,
StG exhibited quantum yields that were much higher
than both 2PyG and 4PVG (Table 1). The values
measured for the single-strand and G-quadruplex
samples containing StG (Èf = 0.33ꢁ0.45) were higher
than the values obtained for duplex DNA
(Èf = 0.20ꢁ0.32). The quantum yields of StG are ꢃ10-
to 100-fold higher than all previously reported fluorescent
guanine mimics in the context of well-folded nucleic acids
with strong base-stacking interactions (53–57).
ÁTm = ꢁ5ꢂC, ꢁ3ꢂC and ꢁ1ꢂC for 2PyG, StG and 4PVG,
respectively (Table 2). Consistent with our results from
hTelo G-quadruplexes, these data suggest that duplex
oligonucleotides containing StG and 4PVG exhibit
duplex-folding equilibria more similar to wild-type hTelo
than either 2PyG or T mutations.
Photophysical properties of 8-substituted guanosines 2–4
Energy transfer properties of modified oligonucleotides
2PyG, StG and 4PVG, exhibit large Stoke’s shifts
(>100 nm) and broad, almost featureless, excitation and
emission spectra (Figures 1B and 2 A–C). Due to their
The excitation spectra of the oligonucleotides containing
8-substituted guanosines exhibit two maxima correspond-
ing to direct excitation (330–400 nm) and indirect excita-
tion via unmodified nucleobases (260 nm). The ratio of
these excitation peaks weighted by the absorbance
properties of the DNA and quantum yields of the probe
can be used to calculate energy transfer efficiencies (ꢀt)
(34,40,41). Here ꢀt is defined as the number of photons
transferred from active nucleobase energy donors to each
energy acceptor, divided by the total number of photons
absorbed by all nucleobases at 260 nm. Due to distance
and geometry constraints, many of the unmodified
nucleobases will be inactive donors, so the reported
efficiencies (Table 1) provide a lower limit for the
transfer efficiencies of the active donors.
Energy transfer efficiencies from unmodified bases to
2PyG, StG and 4PVG were ꢃ2–20-fold higher in
G-quadruplexes (ꢀt = 0.12–0.36) as compared to the
same oligonucleotides in duplex DNA (ꢀt = 0.02–0.06).
Single-stranded samples in Li+ exhibited intermediate
efficiencies (Table 1). While the calculation of energy
transfer efficiencies requires numerous measurements
and data processing, a simple comparison of fluorescence
intensities resulting from DNA excitation versus selective
probe excitation can provide a simple and highly sensitive
readout of G-quadruplex formation. According to this
approach, the intensity of probe fluorescence upon DNA
excitation at 260 nm F(260) is divided by the fluorescence
intensity upon selective probe excitation F(X), where
X = 325 nm for 2PyG, 360 nm for StG and 375 nm for
4PVG. Consistent with the trends in energy transfer
efficiencies (ꢀt), duplex DNA exhibited the lowest
F(260)/F(X) ratios of 0.9–1.4, single-stranded DNA in
Li+ exhibited intermediate F(260)/F(X) ratios of 1.8–2.3,
larger conjugated
p systems, StG and 4PVG are
red shifted by 35–75 nm as compared to 2PyG.
The quantum yields of all three nucleosides (2–4) ap-
proached unity in acetonitrile (È = 0.57–0.74), while
2PyG and 4PVG exhibited much lower quantum yields
in water (È = 0.02 and 0.16). Interestingly, 2PyG and
4PVG exhibited 2-fold higher quantum yields in D2O
than in H2O (Figure 1B). StG, in contrast, exhibited ap-
proximately the same quantum yields in H2O, D2O and
acetonitrile (Figure 1B). Taken together, these results
suggest that water-mediated quenching of 2PyG and
4PVG involves proton transfer between photoexcited
pyridyl nitrogens and bulk solvent. According to molecu-
lar modeling of known structures (49,51), the pyridyl
nitrogen of 2PyG should be partially protected from
bulk solvent upon its incorporation into positions G9
and G23 of hTelo. This may explain the higher quantum
yields of 2PyG in the context of G-quadruplex folded
DNA (34). The same modeling studies suggest that the
pyridyl nitrogen of 4PVG will remain fully solvated in
both quadruplex and duplex DNA. No enhancement in
the quantum yield of 4PVG was therefore predicted upon
its incorporation into DNA.
Quantum yields of 8-substituted guanosines in modified
oligonucleotides
The vast majority of fluorescent guanosine analogs, such
as 2-aminopurine (2AP), 6-methylisoxanthopterin (6-MI)
and 3-methylisoxanthopterin (3-MI) are quenched by
close proximity and collisions with unmodified purine
residues (53,54). These result in large (10–300-fold)

6832 Nucleic Acids Research, 2011, Vol. 39, No. 15
while G-quadruplex structures have the highest F(260)/
F(X) ratios with 3.0–4.7. These values were similar for
all three 8-substituted guanosines, and were independent
of the exact structural features and thermal stabilities of
the G-quadruplexes or duplexes containing them
(Table 1). To evaluate the sensitivity of this F(260)/F(X)
ratiometric analysis, serial 2-fold dilutions of hTeloG9
duplex and G-quadruplex DNA containing StG were per-
formed (Supplementary Figure S7). The ratios in F(260)/
F(X) were concentration independent (quadruplex & 4,
and duplex & 1) and could be readily measured with a
standard fluorimeter and sample holder (1-cm path length)
using 250 pM solutions of StG-labeled DNA
(Supplementary Figure S7). This approach therefore
provides a simple and highly sensitive fluorescence
readout of G-quadruplex folding that is compatible with
extremely diluted sample mixtures.
large losses to intramolecular G-quadruplex thermal
stabilities ranging from 8ꢂC to 24ꢂC, while duplexes con-
taining the same mutations lost only from 2ꢂC to 8ꢂC of
thermal stability (Table 2). On DNA folding and stability,
8-susbtituted guanosines exhibited context-dependent
effects but were generally well-tolerated in both
G-quadruplex and duplex oligonucleotides.
2PyG was the first reported example of a fluorescent
guanine mimic that exhibits the same or higher quantum
yield when base-stacked with neighboring purine residues
(34). As a nucleobase analog, however, 2PyG suffers
from some limitations including a syn glycosidic con-
formational preference, a relatively low-quantum yield
in water (È = 0.02–0.15), and an excitation maximum
in the UV. For these reasons, a second generation
of 8-substituted-20-deoxyguanosines with expanded
pi-systems was developed. One known derivative, 8-
(2-phenylethenyl)-20-deoxyguanosine (StG, Figure 1B)
was previously reported to exhibit an anti glycosidic
conformational preference and the ability to exhibit
photoswitching about the vinyl double bond (39,42,44).
Here, we report a related derivative 8-[2-(pyrid-4-yl)-
ethenyl]-20-deoxyguanosine (4PVG, Figure 1B) that also
exhibits a preference for an anti glycosidic conformation,
but due to the presence of a pyridyl nitrogen atom, it does
not act as a photoswitch. As compared to 2PyG, both of
these vinyl-bridged derivatives exhibit red-shifted excita-
tion and emission maxima, lower perturbations of DNA
structure/stability, and higher quantum yields. To the best
of our knowledge, StG displays the highest quantum yield
of all reported purine base analogs in the context of folded
nucleic acids (È = 0.20–0.45). The quantum yield of StG
is not as environmentally sensitive as 2PyG and 4PVG,
but it can still report DNA folding due to its ability to
serve as an emissive energy acceptor for unmodified
nucleobase donors.
Previous studies have suggested that the enhanced
quantum yields of unmodified guanine residues upon O6
ion coordination are responsible for the unusually efficient
energy transfer reactions mediated by G-quadruplex struc-
tures (34). It was not clear from these results, however, if
efficient energy transfer resulted from the intrinsic
properties of G-quadruplexes, the unusual photophysical
characteristics of 2PyG, or some combination thereof.
Here we report a similar range of transfer efficiencies
for all three 8-substitued guanosines evaluated in
G-quadruplexes (ꢀt = 0.12–0.31), suggesting that efficient
energy transfer results from the intrinsic properties of
G-quadruplex structures, not the probes. In all cases, the
energy transfer efficiencies were structure dependent, with
ꢀt(duplex) < ꢀt(single strand) < ꢀt(G-quadruplex). In the
case of G-quadruplex structures, the combination of effi-
cient energy transfer, high-probe quantum yields, and high
molar extinction coefficient of the oligonucleotide
(e260nm & 250 000 cmꢁ1Mꢁ1) results in very bright fluores-
cence emissions that allow for the detection of
G-quadruplex conformations at oligonuclotide concentra-
tions of 250 pM or lower using a standard fluorimeter and
sample holder. Even in such dilute solutions, a simple
comparison of emission intensities resulting from selective
probe excitation versus excitation of the DNA provides a
CONCLUSIONS
Fluorescence phenomena enable powerful and readily ac-
cessible technologies for probing biomolecule folding and
activity (26). Fluorescence-based assays involving fluores-
cent nucleobase analogs typically assess conformational
changes using probes that exhibit lower quantum yields
when base-stacked with neighboring residues (58–62).
Probes that remain highly emissive when involved in
base-stacking interactions will increase the sensitivity of
nucleic acid folding studies, and may provide a means to
differentiate DNA/RNA folds where alternative
base-stacking interactions are present (eg. duplex versus
quadruplex). Here, we demonstrate how highly emissive
probes that mimic guanine residues can be used to differ-
entiate DNA secondary structures by estimating energy
transfer efficiencies between unmodified nucleobases and
8-substituted guanines. To the best of our knowledge, this
represents a new paradigm in detecting conformational
changes in nucleic acids. While FRET-based studies
using large ‘external’ probes are frequently reported (21),
interpretations of the resulting data are limited to changes
in probe-to-probe distances, whereas the use of an
‘internal’ fluorescent base analog can provide a direct
readout of the characteristic photophysical properties of
secondary structures of nucleic acids containing them.
Notably, this can be accomplished by introducing a very
small modification (e.g. a single styrene or pyridine group)
into the oligonucleotide at a strategic location to provide a
highly sensitive and minimally perturbing fluorescent
readout of structure.
Polymorphic G-quadruplex structures derived from the
human telomeric repeat (hTelo) provide demanding model
systems to evaluate internal fluorescent probes for their
potential impact on DNA folding and stability. To effect-
ively mimic guanosine in G-quadruplexes, both the
Watson–Crick and Hoogsteen hydrogen bonding faces
must be maintained. Previous studies have demonstrated
that subtle changes to either face (e.g. 7-deazaguanine,
6-thioguanine or inosine) result in large losses in
G-quadruplex folding kinetics and stability (63). Indeed,
single base mutations (G!T) in hTelo DNA resulted in

Nucleic Acids Research, 2011, Vol. 39, No. 15 6833
13. Phan,A.T., Luu,K.N. and Patel,D.J. (2006) Different loop
arrangements of intramolecular human telomeric (3+1)
G-quadruplexes in K+ solution. Nucleic Acids Res., 34,
5715–5719.
highly convenient, sensitive and reliable means to charac-
terize the folded states of oligonucleotides. The trends
from these ratiometric analyses matched those from
detailed analyses of energy transfer efficiencies. In both
cases, the trends were independent of the exact structural
features of the G-quadruplex or 8-substituted guanosine
probe used. Given the simplicity and sensitivity of this
approach, we expect that it will be compatible with con-
formational analyses using fluorescence microscopy and
single molecule spectroscopy.
14. Ambrus,A., Chen,D., Dai,J.X., Jones,R.A. and Yang,D.Z. (2005)
Solution structure of the biologically relevant G-quadruplex
element in the human c-MYC promoter. implications for
g-quadruplex stabilization. Biochemistry, 44, 2048–2058.
15. Phan,A.T., Kuryavyi,V., Burge,S., Neidle,S. and Patel,D.J. (2007)
Structure of an unprecedented G-quadruplex scaffold in the
human c-kit promoter. J. Am. Chem. Soc., 129, 4386–4392.
16. Jin,R.Z., Gaffney,B.L., Wang,C., Jones,R.A. and Breslauer,K.J.
(1992) Thermodynamics and structure of a DNA tetraplex - a
spectroscopic and calorimetric study of the tetramolecular
complexes of d(TG3T) and d(TG3T2G3T). Proc. Natl
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SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
17. Mergny,J.L., Phan,A.T. and Lacroix,L. (1998) Following
G-quartet formation by UV-spectroscopy. FEBS Lett., 435,
74–78.
18. Phan,A.T. and Mergny,J.L. (2002) Human telomeric DNA:
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19. Parkinson,G.N., Lee,M.P.H. and Neidle,S. (2002) Crystal
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ACKNOWLEDGEMENTS
We are grateful to Professor Andrea Vasella, Dr Fabio
De Giacomo and Dr Thomas Steinlin for providing
8-substituted guanines for initial fluorescence studies (64).
20. He,F., Tang,Y.L., Wang,S., Li,Y.L. and Zhu,D.B. (2005)
Fluorescent amplifying recognition for DNA G-quadruplex
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Takenaka,S. (2006) G quadruplex-based FRET probes with the
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efficient fluorometric detection of the potassium ion.
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23. Schaffitzel,C., Berger,I., Postberg,J., Hanes,J., Lipps,H.J. and
Pluckthun,A. (2001) In vitro generated antibodies specific for
telomeric guanine-quadruplex DNA react with Stylonychia lemnae
macronuclei. Proc. Natl Acad. Sci. USA, 98, 8572–8577.
24. Alzeer,J. and Luedtke,N.W. (2010) pH-mediated fluorescence and
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FUNDING
University of Zurich Forschungskredit (57131901); Swiss
National Science Foundation (130074). Funding for open
access charge: Swiss Federal Grant.
Conflict of interest statement. None declared.
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