Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA

Article abstract of DOI:10.1093/nar/gkr010

To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A^T) and its photophysical characteri

Full text of DOI:10.1093/nar/gkr010

Published online 29 January 2011
Nucleic Acids Research, 2011, Vol. 39, No. 10 4513–4524
doi:10.1093/nar/gkr010
Characterization of photophysical and
base-mimicking properties of a novel
fluorescent adenine analogue in DNA
1
2
Anke Dierckx , Peter Diner , Afaf H. El-Sagheer3,4, Joshi Dhruval Kumar1,
´
Tom Brown3, Morten Grøtli2 and L. Marcus Wilhelmsson1,*
1Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology,
2Department of Chemistry, Medicinal Chemistry, University of Gothenburg, S-41296 Gothenburg, Sweden,
4
3School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK and Department of
Science and Mathematics, Chemistry Branch, Faculty of Petroleum and Mining Engineering, Suez Canal
University, Suez 43721, Egypt
Received November 12, 2010; Revised January 3, 2011; Accepted January 4, 2011
ABSTRACT
emissive molecules which are similar in shape to natural
nucleobases and are able to form one or more hydrogen
bonds to a natural nucleobase in the complementary
strand. This means that these artificial bases do not rad-
ically alter the structure of DNA, and therefore they have
major advantages compared to other dyes which are co-
valently tethered to the DNA outside the base-stack (e.g.
Cy-dyes, fluorescein, Alexa dyes or rhodamines).
Importantly, fluorescent base analogues allow experi-
ments to be performed while preserving the native struc-
ture of DNA by avoiding bulky external probes.
Furthermore, they are located rigidly in the DNA
base-stack, in contrast to the covalently attached dyes,
and this restricted movement results in a more predictable
orientation which can therefore yield more reliable fluor-
escence resonance energy transfer (FRET) data and fluor-
escence anisotropy data. These probes have therefore
become increasingly popular over the past decade for
studying interactions between DNA/RNA and other mol-
ecules and to explore nucleic acid structure.
Over the past years various classes of base analogues
have been developed, each with specific properties. With
exception of the tricyclic cytosine family described in more
detail below, virtually all these classes show significant
sensitivity to their microenvironment. The pyrimidine ana-
logues designed by Tor and co-workers (2–4), pyrrolo-dC
(5), the blue fluorescent triazole deoxycytidine analogues
(6) and the base discriminating fluorescent bases (BDF)
designed by Saito and co-workers (7–11) are just a few
examples of this expanding class of molecules.
Furthermore, the pteridine analogues of guanine (3-MI
and 6-MI) (12–15) and adenine (6-MAP and DMAP)
(16) have also been studied thoroughly. Other interesting
purine analogues are the adenine analogues A-3CPh and
To increase the diversity of fluorescent base
analogues with improved properties, we here
present the straightforward click-chemistry-based
synthesis of a novel fluorescent adenine-analogue
triazole adenine (AT) and its photophysical charac-
terization inside DNA. AT shows promising
properties compared to the widely used adenine
analogue 2-aminopurine. Quantum yields reach
>20% and >5% in single- and double-stranded
DNA, respectively, and show dependence on neigh-
bouring bases. Moreover, AT shows only a minor
destabilization of DNA duplexes, comparable to
2-aminopurine, and circular dichroism investiga-
tions suggest that AT only causes minimal structural
perturbations to normal B-DNA. Furthermore, we
find that AT shows favourable base-pairing
properties with thymine and more surprisingly also
with normal adenine. In conclusion, AT shows strong
potential as a new fluorescent adenine analogue
for monitoring changes within its microenvironment
in DNA.
INTRODUCTION
Fluorescence is a very powerful and commonly used tech-
nique for the study of macromolecules, such as DNA,
RNA and proteins (1). The virtually non-fluorescent
nature of the regular nucleic acid bases makes artificial
fluorophores such as fluorescent base analogues important
and excellent tools in investigations of DNA or RNA
systems. Fluorescent base analogues are significantly
*To whom correspondence should be addressed. Tel: +46 31 772 3051; Fax: +46 31 772 3858; Email: marcus.wilhelmsson@chalmers.se
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/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

4514 Nucleic Acids Research, 2011, Vol. 39, No. 10
A-4CPh that show an increased emission inside RNA
(17,18) and the 7-deazapurines (19,20). Recently, our
group has developed and extensively characterized a
class of tricyclic cytosine analogues, tC, tCo and tCnitro
.
Surprisingly, neither tC nor tCo show sensitivity to their
environment in duplex DNA. Lately, we also presented
tCo and the non-emissive tCnitro as the first nucleobase
FRET-pair, yielding accurate distance and orientation in-
formation in DNA (21–23). [for recent reviews on fluor-
escent (nucleic acid base) analogues, see references (24),
(25) and (1)].
One of the most widely used fluorescent base analogues
is the adenine analogue 2-aminopurine, which shows a
quantum yield of 0.68 in water. However, this decreases
dramatically in both single- (0.007–0.02) and double-
stranded DNA (0.0007–0.01) (26). Moreover, a destabil-
ization of the duplex of 10ꢀC has been determined using
melting studies (27). Furthermore, 2-aminopurine is
capable of forming base pairs with both thymine and
cytosine and is, thus, not selective (28,29). Nonetheless,
it has found use in countless studies as discussed further
below.
Fluorescent nucleic acid base analogues have found
many applications over the years, of which only a few
will be highlighted here [for a broader overview see
(1,24,30,31)]. The future perspective of personalized
medicine is to allow treatment based on individual
genetic data. With this objective in mind, the detection
of single nucleotide polymorphisms (SNP) has gained
increasing attention and fluorescent base analogues, such
as the class of BDF analogues developed by Saito and
co-workers have been designed for this purpose. The prin-
ciple is based on these analogues showing a distinct dif-
ference in emissive signal depending on the opposite base
(7–10). Furthermore, many analogues are recognized as
substrates by DNA/RNA polymerases and have conse-
quently been used in studies concerning the enzyme mech-
anism, kinetics, substrate specificity and fidelity (5,32–38).
2-Aminopurine has been used in numerous studies on T4
DNA polymerase and Klenow fragment binding to
primer-template DNA as well as T7 RNA polymerase
interaction with the DNA promotor (39–44). Also, in
studies on the mechanism of other proteins and their
binding to RNA or DNA, fluorescent base analogues
have been proven to be highly useful. An excellent
example of such an application for DNA is the role of
3-MI in an investigation of the dissociation of the
multimeric form of the HIV-1 integrase complex upon
DNA binding (45). In the RNA-context, protein binding
studies have also been performed using fluorescent base
analogues, for example, 2-aminopurine has been utilized
in studies of adenosine deaminases (ADARs) which play a
role in RNA editing of eukaryotic pre-mRNA (46,47).
Finally, fluorescent base analogues have recently entered
the area of DNA nanotechnology, exemplified by the use
of tCo in monitoring the local stability of self-assembling
DNA hexagons (48).
Figure 1. Structure of 8-(1-pentyl-1H-1,2,3-triazole-4-yl)-20-deoxyadeno-
sine, AT, in which the triazole ring with n-pentyl, added to the normal
adenine structure, is shown in grey.
compared to other fluorescent adenine analogues such as
the commercially available 2-aminopurine. In a previous
investigation, several adenosine derivatives of AT were
studied and shown to exhibit a very high-quantum yield
in THF and red-shifted absorption relative to the DNA
absorption band. One of the compounds, having an
isopentyl substituent in the triazole ring, showed a very
high-quantum yield in both THF (0.62) and water (>0.50)
(49). In this work, we characterize the 20-deoxyadenosine
form of AT both in methanol and water. In addition, we
have incorporated it into 10 different 10-mer oligonucleo-
tides with various base-stacking environments in order to
perform a photophysical and structural characterization
of AT in DNA. We find that AT causes only minor per-
turbations to natural B-DNA. Furthermore, quantum
yields for AT incorporated into single- and
double-stranded DNA reach maximum values that far
exceed corresponding values for 2-aminopurine.
Moreover, we have investigated the base-pairing specifi-
city of AT with all four natural DNA-bases. To our
surprise, we find that AT forms equally stable base pairs
with thymine and adenine. In conclusion, AT is a very
promising new fluorescent base analogue, showing high
sensitivity to its microenvironment.
MATERIALS AND METHODS
The synthesis of 8-(1-pentyl-1H-1,2,3-triazole-4-yl)-20-
deoxyadenosine and of the triazolyl 20-deoxyadenosine
phosphoramidite monomer (see Scheme 1 in ‘Results’
section) as well as its incorporation into oligonucleotides
and purification of the oligonucleotide strands are
described in the Supplementary Data S1.
Sample preparation
All samples used in this work, unless stated otherwise,
were prepared in a sodium phosphate buffer, pH 7.5
with a total salt concentration of 500 mM. The oligo-
nucleotide concentration was determined by measuring
the absorption at 260 nm. The extinction coefficients of
the modified oligonucleotide single strands (sequences
listed in Table 1) were calculated by summation of the
extinction coefficients of the natural oligonucleotides
and of the AT-monomer. This sum was multiplied by 0.9
Here we report the synthesis and photophysical charac-
terization of a new fluorescent adenine analogue in DNA,
8-(1-pentyl-1H-1,2,3-triazole-4-yl)-20-deoxyadenosine
(AT) (Figure 1), which shows promising features

Nucleic Acids Research, 2011, Vol. 39, No. 10 4515
Table 1. DNA melting temperatures of the 10 AT-modified
DNA-duplexes (TmAT), the corresponding unmodified duplexes (Tm
and the difference in melting temperature between them (ꢀTm)
A
)
AT
Tm
(ꢀC)c
A
DNA sequencea
Neighbouring
bases = name
of oligomerb
Tm
(ꢀC)c
ꢀTm
(ꢀC)
50-d(CGCACATATCG)-30
50-d(CGCAGATGTCG)-30
50-d(CGCAAATATCG)-30
50-d(CGCATATATCG)-30
50-d(CGCATATGTCG)-30
50-d(CGCAGATATCG)-30
50-d(CGCACATTTCG)-30
50-d(CGCAAATCTCG)-30
50-d(CGCACATCTCG)-30
50-d(CGCAGATCTCG)-30
CA
GG
AA
TA
TG
GA
CT
AC
CC
GC
43
45
41
39
42
43
45
46
49
49
52
54
50
47
50
50
52
53
56
54
ꢁ9
ꢁ9
ꢁ9
ꢁ8
ꢁ8
ꢁ7
ꢁ7
ꢁ7
ꢁ7
ꢁ5
aFor the unmodified strands AT is replaced by A.
bPurines neighbouring AT are shown in bold and pyrimidines are
shown in italic.
cSamples were prepared in phosphate buffer (500 mM Na+, pH 7.5) at a
duplex concentration of 2.5 mM.
M
ꢁ1cmꢁ1 and eTG = 90 400 Mꢁ1cmꢁ1. Extinction coeffi-
cients of the unmodified oligonucleotides were calculated
in the same way.
UV melting experiments
Equal volumes of 5 mM solutions of single-stranded oligo-
nucleotides in buffer were mixed at room temperature. In
order to achieve a correct and complete hybridization the
samples were heated to 85ꢀC, followed by cooling to 5ꢀC
at the rate of 1ꢀC/min. Next, data were recorded during
heating of the samples to 85ꢀC, followed by cooling to 5ꢀC
at a rate of 0.5ꢀC/min. The temperature was kept at 85ꢀC
for 5 min between heating and cooling. For unmodified
duplexes a temperature range of 15ꢀC to 92ꢀC was used
for recording those data. The melting curves were
obtained on a Varian Cary 4000 spectrophotometer with
a programmable multi-cell temperature block using an ab-
sorption wavelength of 260 nm. Melting temperatures pre-
sented in this article are averages of the temperature values
at the maximum of the first derivative and at half
maximum of the melting curves and were measured at
least twice.
Scheme 1. (a) Br2/NaOAc-buffer, r.t., overnight, 81%. (b) Pd(PPh3)4
(4 mol%), CuI (8 mol%), TMS-acetylene, Amberlite IRA-67, THF,
40ꢀC, 2 h, 91%. (c) (i) DMTrCl, pyridine, overnight, r.t.; (ii) NH3
(25% aq.), 2 h 65%. (d) (i) Pentylbromide, NaN3, water, 140ꢀC, 1 h;
(ii) CuI, ethyl acetate, 14 h, r.t., 76%. (e) (i) TMSCl, pyridine, 2 h;
(ii) BzCl, 3 h; (iii) Water, 15 min then NH3 (aq.), 30 min, 68%.
(f) DIPEA (0.11 ml, 0.64 mmol), 2-Cyanoethyl-N,N-diisopropylchloro-
phosphoramidite, DCM, 1 h, 91%. (g) Acetic acid, 30 min, 34%.
Circular dichroism
to correct for base-stacking interactions. The extinction
coefficient of the highly soluble AT was determined by
dissolving 1.4 mg of compound in 50 ml of methanol. An
absorption shift of 4 nm was observed compared to AT in
Milli-Q water. The extinction coefficient of AT in water
was therefore calculated using the absorption value at
264 nm in methanol (9400 Mꢁ1cmꢁ1). The extinction coef-
ficients that were used for the different bases at 260 nm
Circular dichroism (CD) spectra were recorded on a
Chirascan CD spectrophotometer at 25ꢀC. Spectra of all
samples were recorded between 200 and 350 nm and cor-
rected for background contributions. Spectra of solutions
containing 2.5 mM DNA duplexes, prepared as described
above, were recorded and data were averaged over at least
five measurements at a scan rate of 2 nm/s.
were
eG = 11 800 Mꢁ1cmꢁ1
AT = 9400 Mꢁ1cmꢁ1. Using these values, the following
extinction coefficients were obtained for the modified
oligonucleotides: CT = 86 500
GA = 95 900 Mꢁ1cmꢁ1
ꢁ1cmꢁ1, eGC = 88 800 Mꢁ1cmꢁ1, eCA =91 900 Mꢁ1cmꢁ1
GG = 92 700 Mꢁ1cmꢁ1, eCC = 84 800 Mꢁ1cmꢁ1, eTA
93 600 Mꢁ1cmꢁ1 AA = 99 000 Mꢁ1cmꢁ1
AC = 91 900
eT = 9300 Mꢁ1cmꢁ1
eA = 15 300 Mꢁ1cmꢁ1
,
eC = 7400 Mꢁ1cmꢁ1
,
Steady-state fluorescence measurements
,
and
Quantum yields (Ff) of the AT-monomer and the different
AT-modified oligonucleotides were determined relative to
the quantum yield of quinine sulphate (Ff = 0.55) in 0.5 M
H2SO4 at 25ꢀC (50). Samples containing duplex DNA
were prepared as described above. The monomer or
samples containing single-stranded oligonucleotides were
e
e
, e
M
,
e
=
,
e
, e

4516 Nucleic Acids Research, 2011, Vol. 39, No. 10
set to an absorption of ꢂ0.03 at the excitation wavelength.
three-exponential expressions with the program Fluofit
Pro v.4 (PicoQuant GmbH).
Spectra were recorded on
a
SPEX fluorolog
3
spectrofluorimeter (JY Horiba). For the duplexes and
single-stranded oligonucleotides an excitation wavelength
of 300 nm was used and emission spectra were recorded
between 305 and 550 nm. The monomer was excited at
282 nm and the emission was monitored between 290
and 700 nm. For the reference, quinine sulphate,
emission was recorded between 305 and 700 nm.
Steady-state excitation anisotropy spectra were
recorded on a SPEX fluorolog 3 spectrofluorimeter (JY
Horiba) using Glan polarizers and emission was
measured at 355 nm using excitation wavelengths from
280 to 340 nm. The polarized excitation spectra of the
AT-monomer were recorded between 240 and 350 nm in
a H2O:ethylene glycol (1:2 mixture) glass at ꢁ100ꢀC,
monitoring the emission at 352 nm. The fluorescence an-
isotropy was calculated as:
Computation of the internal wobble of AT in DNA
Duplex samples of strands AA and TA (Table 1) were
prepared as described above in phosphate buffer
(500 mM Na+, pH 7.5) containing 65% (w/v) (ꢁ/ꢁwater
&
20) and 77.2% (w/v) sucrose (ꢁ/ꢁwater & 58). An excess of
12% of the unmodified strand was used. Samples were
annealed by heating to 75ꢀC for 20 min, followed by
slow cooling to room temperature. Steady-state excitation
anisotropy measurements were performed as described
above for which spectra were averaged over 22 repeats
and were corrected for background contributions.
Internal dye motion was computed using an in-house
designed Matlab program (S. Preus, personal communica-
tion) based on calculations carried out by Barkley and
Zimm (51).
IVV ꢁ GIVH
r ¼
IVV+2 G IVH
RESULTS
Synthesis of AT phosphoramidite monomer
where the instrumental correction factor
Starting from commercially available 20-deoxyadenosine
IHV
G ¼
1, 8-bromoadenosine
2 was easily obtained using
IHH
bromine in a sodium acetate buffer (Scheme 1) as previ-
ously described (52). The TMS-protected alkyne 3 was
installed in the 8-position in good yield (91%) via a
standard Sonogashira coupling protocol (53). The
50-hydroxy group was protected using 4,40-dimethoxytrityl
chloride in dry pyridine, followed by an in situ
deprotection of the TMS-group using aqueous ammonia
(25%) yielding compound 4 in 65%. In the next step, the
pentyl azide was generated in situ through the reaction
between pentyl bromide and sodium azide, followed by
the addition of compound 4 and copper iodide, yielding
the cycloaddition product 5 in 76% yield. A small sample
of compound 5 was treated with acetic acid to generate
sufficient amount of 8 for measuring its fluorescence
quantum yield and anisotropy. The exocyclic amino
group of compound 5 was protected with a benzoyl
group. In the last step, compound 7 was prepared using
DIPEA and 2-cyanoethyl-N,N-diisopropylchloropho-
sphoramidite in dichloromethane (91%).
and IXY is the excitation spectrum for which the subscripts
X and Y denote polarization directions of the excitation
and emission light, respectively, and H and V refers to
horizontal and vertical, respectively. For an immobile
fluorophore, such as AT in H2O/ethylene glycol glass,
the fundamental anisotropy (r0i) for a certain transition
(i) is related to the angle between the absorbing and the
emitting transition moments as follows:
À
Á
1
r0i ¼ 3 cos2 ꢀi ꢁ 1 :
5
Time-resolved fluorescence measurements
Fluorescence
lifetimes
were
measured
using
time-correlated single-photon counting. The excitation
pulse was generated with a Tsunami Ti:sapphire laser
(Spectra-physics) that was pumped by a Millenia Pro X
laser (Spectra-Physics). The Tsunami output was tuned to
900 nm and frequency-tripled to 300 nm. Samples were
excited with a repetition rate of 80 MHz, a 1-2 ps pulse
width, IRF 70 ps (FWHM) and a time-window of 10 ns.
However, for the AT monomer and the single stranded
samples AA and GC (Table 1) the frequency was
reduced to 4 MHz by a pulse picker (Spectra physics,
Model 3985) and a time window of 20 ns was used. The
emission was monitored at 355 nm and photons were col-
lected by a microchannel-plate photomultiplier tube
(MCP-PMT R3809U-50; Hamamatsu). These counts
Spectroscopic characterization of the AT monomer
The absorption and emission spectra of AT, where the
triazole-modified A-nucleobase constitutes the chromo-
phoric/fluorophoric unit of the nucleoside, in Milli-Q
water are shown in Figure 2. Their shapes are virtually
identical to the corresponding spectra recorded for AT in
methanol (data not shown). In water, the lowest energy
absorption band is centred at 282 nm with an extinction
coefficient of 16 500 Mꢁ1cmꢁ1 and the emission maximum
is located at 353 nm. The absorption spectrum recorded
for AT in methanol shows a redshift of 4 nm compared to
in water whereas a blueshift of 7 nm was observed for the
corresponding emission spectrum of AT. Moreover, AT
shows a very high quantum yield both in water (0.61)
and in methanol (0.49). Furthermore, a lifetime of 1.8 ns
were fed into
a multichannel analyzer with 4096
channels (Lifespec, Edinburgh Analytical Instruments),
where a minimum of 10 000 counts were recorded in the
top channel. The intensity data were convoluted with the
instrument response function and fitted to one-, two- or

Nucleic Acids Research, 2011, Vol. 39, No. 10 4517
Figure 4. CD spectra of all 10 DNA duplexes containing the AT
analogue. Duplexes are denoted by the bases neighbouring AT and
consist of the modified strands GA (black), CT (red), GC (dark
blue), CA (brown), GG (purple), CC (orange), TA (light blue), AA
(grey), AC (pink) and TG (green) hybridized to the complementary
natural DNA strand. Spectra were recorded in phosphate buffer
(500 mM Na+, pH 7.5) at 25ꢀC at a duplex concentration of 2.5 mM.
Figure 2. Absorption (dashed line) and emission (solid line) spectrum
of the AT monomer in water.
combinations of purines and pyrimidines and their prox-
imity both 30 and 50 to AT. The melting temperatures of
unmodified and modified duplexes prepared by mixing
with their unmodified complementary DNA strands are
presented in Table 1. Comparison between the melting
temperatures of the modified and the corresponding un-
modified duplexes shows an average drop in melting tem-
perature of 8ꢀC, and thus a destabilization of double
stranded DNA upon incorporation of AT. Further
analysis of the changes in Tm suggests that sequences
with purines 30 of AT have the largest destabilizing effect
whereas pyrimidines at the 30-side of AT generally have a
smaller effect.
Figure 3. Excitation anisotropy spectrum (r0, solid line) of the AT nu-
cleoside in a H2O/ethylene glycol glass (1:2 mixture) at ꢁ100ꢀC. The
isotropic absorption (Aiso, dashed line) is shown as a comparison and
was measured in Milli-Q water.
Circular dichroism (CD). The CD spectra that were
recorded for the duplexes, consisting of a modified
strand containing one AT (Table 1) and its unmodified
complementary sequence are presented in Figure 4.
Analysis of the spectra between 200 and 300 nm shows a
high resemblance to the CD signature of a natural
B-DNA-helix, which is characterized by a positive band
at 275 nm, a negative band at 240 nm, a band which is less
negative or positive at 220 nm and a narrow negative band
in the region between 220 and 190 nm, preceded by a large
positive peak at 180–190 nm. Some modified duplexes
show an almost identical CD-spectrum to their natural
DNA (CT, CA, TG, CC and AA) (Supplementary Data
S2), whereas others show more distinct differences
between the corresponding spectra (strands GA, GC,
GG, TA and AC) (Supplementary Data S2). Sequences
GA, GC and GG, having a guanine flanking AT at the
50-side, show the most perturbed CD signals. As an illus-
tration of this variation, one of the latter spectra (GG) is
shown in Figure 5 and compared to the CD spectrum of
the corresponding unmodified helix and to the absorption
difference between the modified and natural duplex of this
sequence. The resulting differential absorption band cor-
relates well to the regions (ꢂ210, 230 and 295 nm) where
was recorded for the AT monomer in water. A small
residual could be observed, which is most probably a
result of small deviations in the instrument response
function.
The fundamental fluorescence anisotropy of AT (r0,
Figure 3) was measured in a H2O/ethylene glycol glass
(1:2 mixture). A constant value of r0 = 0.38 can be
observed between 280 and 320 nm, which approaches the
theoretical maximum value of 0.4 and suggests that
the emission and absorption transition moments are vir-
tually parallel in this region. The constant r0 value in this
region also indicates a single transition dipole moment in
this absorption band.
Structure and stability of DNA duplexes containing AT
UV duplex melting experiments. The 10 modified and
normal single-stranded DNA sequences that were used
in this study are listed in Table 1. To examine the influence
of various bases surrounding the modified adenine AT, the
neighbouring positions are varied in these 10 sequences.
Sequences were designed in order to evaluate

4518 Nucleic Acids Research, 2011, Vol. 39, No. 10
the CD-spectra of natural and modified duplexes differ
most significantly (Figure 5).
When a mismatched guanine or cytosine is positioned
opposite to AT, further destabilization of the modified
duplexes by an average of 8ꢀC can be observed.
Surprisingly, the positioning of an adenine opposite of
AT results in virtually no change in melting temperatures
(<1ꢀC) compared to the duplexes with the thymine-AT
base pair. In contrast, an adenine–adenine mismatch in
the three unmodified duplexes leads to an average drop
in melting temperature of 14ꢀC in comparison to the
natural adenine–thymine pair (data not shown). This
suggests that AT is capable of forming equally stable
base pairs with thymine and adenine.
The quantum yields of the duplexes containing a
guanine-AT mismatch increase between 13 and 36% in
comparison to the normal matched modified duplexes
and the corresponding values for cytosine-AT mismatches
are 0 and 36%. For the adenine-AT base pair a decrease in
quantum yield between 0 and 27% is observed. However,
we calculated up to 16% of the duplexes to be denatured
at the temperature at which these measurements were per-
formed (25ꢀC). Taking into account this and experimental
errors when measuring low-quantum yields, the changes
are fairly low and should not be over-interpreted.
Furthermore, when comparing the spectral shape and
emission maxima as well as CD-spectra of the various
mismatched duplexes to that of the corresponding
matched duplexes, virtually no changes can be observed
(data not shown).
Base pairing specificity. To investigate the ability of AT to
form base pairs with bases other than thymine, we chose
three of the modified duplexes and replaced the base
opposite AT by a guanine, cytosine and adenine, respect-
ively (Table 2). These three sequence contexts were chosen
so that the influence of neighbouring bases could also be
explored: AT is surrounded either by (i) two purines,
(ii) two pyrimidines or (iii) a purine and a pyrimidine.
The melting temperatures and the quantum yields that
were obtained for these mismatched duplexes are listed
in Table 2. For comparison, the data recorded for the
modified matched duplexes are also shown. The neigh-
bouring bases did not significantly influence the depres-
sion in melting temperature caused by the mismatches.
Computation of the internal wobble of AT in DNA. To
estimate the internal motion of AT inside the base stack
of DNA, anisotropy measurements were recorded for two
of the modified duplexes, AA and TA (Table 1), which
were prepared in phosphate buffer (500 mM Na+, pH
7.5) of high viscosity (65 and 77.2% sucrose w/v). The
high viscosity hinders motion of the DNA helices in
between the moment of excitation and emission. In
standard phosphate buffer (500 mM Na+, pH 7.5) anisot-
ropy values of 0.30 and 0.21 were recorded for samples AA
and TA, respectively. When performing the same
Figure 5. CD spectra of the modified GG-duplex (solid line) and of the
corresponding natural duplex (dashed line). The difference in absorp-
tion spectra of AT and A (dotted line) was obtained by subtracting the
absorption spectra of equimolar solutions of the natural GG-duplex
from the modified GG-duplex. Spectra were recorded in phosphate
buffer (500 mM Na+, pH 7.5) at 25ꢀC at a duplex concentration of
2.5 mM.
Table 2. Comparison of the melting temperatures and quantum yields of duplexes containing a mismatched base opposite of AT with its
matched counterparts
T
AT
AT
b,c
Neighbouring
bases = name of
Base
opposite
Tm
Tm
ꢀTm
(ꢁꢀC)
ꢁf
ꢁfA b,c
mismatch
mismatch
(ꢁꢀC)b
(ꢁꢀC)b
oligonucleotidea
ATa
GA
CT
CA
G
C
A
G
C
A
G
C
A
36
36
43
36
36
46
36
36
42
ꢁ7
ꢁ7
0
ꢁ9
ꢁ9
1
ꢁ7
ꢁ7
ꢁ1
0.009
0.010
0.008
0.006
0.005
0.004
0.015
0.015
0.008
0.008
0.005
0.011
43
45
43
aOligonucleotides are named using the two bases neighbouring AT. Full sequences can be found in Table 1. The base opposite AT in the duplexes is
replaced by the three other bases that normally do not base-pair with adenine (G, C, A).
bSamples were prepared in phosphate buffer (500 mM Na+, pH 7.5) at a duplex concentration of 2.5 mM.
cFluorescence quantum yields were determined relative to the reference quinine sulphate in 0.5 M H2SO4 (ꢁf = 0.55) and are averages of at least two
independent measurements.

Nucleic Acids Research, 2011, Vol. 39, No. 10 4519
Table 3. Wavelengths of emission maxima and quantum yields of the
ten AT-modified single strands
measurements in high-viscosity buffer, a limiting anisot-
ropy of on average 0.34 was measured for both samples,
AA and TA. The difference between the values measured
at 65 and 77.2% sucrose (w/v) was within experimental
error. The limiting anisotropy recorded here is lower than
the fundamental anisotropy that was recorded for the AT
monomer in a vitrified matrix (0.38). The difference in the
anisotropy in DNA in viscous solution and the monomer
in a vitrified matrix is a result of motions of AT inside the
base-stack and calculations (51) suggest an internal
wobble of 16 degrees.
Oligonucleotidea
Emmax (nm)
Ffb
AA
CA
TA
GC
AC
CC
GA
CT
TG
GG
353
351
352
358
357
355
353
358
355
355
0.21
0.090
0.076
0.042
0.031
0.021
0.018
0.014
0.006
0.005
aOligonucleotides are named by the two bases neighbouring AT. Full
sequences can be found in Table 1.
Photophysical characterization of AT in DNA context
bFluorescence quantum yields were determined relative to the reference
quinine sulpfate in 0.5 M H2SO4 (ꢁf = 0.55) at an excitation wave-
length of 300 nm and are averages of three independent measurements.
Samples were prepared in phosphate buffer (500 mM Na+, pH 7.5).
Fluorescence properties of AT in single-stranded DNA. The
fluorescence properties of AT upon incorporation into the
10 single-stranded oligonucleotides (listed in Table 1) are
shown in Table 3. These data show no large shift in the
emission maxima (351–358 nm) in comparison to the free
AT-monomer (354 nm). Furthermore, when comparing
the emission spectra of the various modified oligonucleo-
tides to that of the monomer AT and the corresponding
modified duplex, only minimal changes in the spectral
envelope can be observed (data not shown).
Table 4. Wavelength of emission maxima and quantum yields of the
ten AT-modified double-strands
Oligonucleotidesa
Emmax (nm)
ꢁfb
AA
TA
CA
AC
GA
CT
GC
CC
TG
GG
350
353
354
349
351
351
351
352
351
354
0.050
0.016
0.011
0.009
0.008
0.005
0.005
0.004
0.003
0.003
The fluorescence quantum yields for the 10 modified
oligonucleotides range between 0.005 and 0.21 (Table 3).
The AA sequence shows the highest quantum yield (0.21).
In general, with exception of GA, the highest quantum
yields are observed for the sequences with an adenine
flanking AT at the 30-side (AA, CA, TA). Lower
quantum yields are observed for a cytosine 30 of AT
(GC, AC, CC) and these values decrease further for
having a thymine (CT) and finally a guanine 30 of AT
(TG, GG). Not surprisingly, the lowest quantum yield
(0.005) was found for the sequence with two guanines
(GG) neighbouring AT. The average quantum yield
(0.051) is approximately 12 times lower for the modified
oligonucleotides in comparison to the free AT-monomer
(0.61).
aOligonucleotides are named by the two bases neighbouring AT. Full
sequences can be found in Table 1.
bFluorescence quantum yields were determined relative to the reference
quinine sulphate in 0.5 M H2SO4 (ꢁf = 0.55) and are averages of at
least two independent measurements. Samples were prepared in phos-
phate buffer (500 mM Na+, pH 7.5) at a duplex concentration of
2.5 mM.
Average lifetimes of AT incorporated into the 10
modified oligonucleotides listed in Table
1 range
single-stranded oligonucleotides, sequence AA shows the
highest quantum yield. Furthermore, the observation that
an adenine 30 of AT (AA, TA, CA) yields the highest fluor-
escence (with exception of GA) and a guanine 30 of AT
(TG, GG) yields the lowest quantum yield is found also for
the duplex case. As for the single strands, sequence GG
shows the lowest quantum yield in the duplex. The
average quantum yield of AT incorporated into duplexes
(0.011) is reduced ꢂ5-fold in comparison to the average
quantum yield of the single strands (0.051).
between 0.9 and 2.0 ns. Fluorescence intensity decays
were fitted to three lifetimes for all samples, except for
strands AA and GC, which showed a mono-exponential
decay. For all strands, except for GC (ꢂ = 2.0 ns), the
average lifetime of the incorporated AT is lower than the
lifetime of the AT monomer in water (1.8 ns).
Fluorescence properties of AT in double-stranded
DNA. The fluorescence properties of the AT-monomer
when incorporated into duplex DNA, consisting of
the modified sequences (listed in Table 1) with their
unmodified complement are shown in Table 4. The
Average lifetimes of AT incorporated into the 10
modified duplexes vary between 0.2 and 1.2 ns. All fluor-
escence intensity decays for these samples were fitted to
three fluorescence lifetimes. For all duplexes, the average
lifetimes of AT are lower than the lifetime of the AT
monomer in water. Also when compared to the corres-
ponding lifetimes of AT in single-stranded oligonucleo-
tides, these lifetime values are lower except for strands
GG, TG and GA.
emission
maxima
for
the
modified
duplexes
(349–354 nm) are virtually not shifted in comparison to
the AT-monomer (354 nm) and the modified single
strands (351–358 nm).
The fluorescence quantum yields that were measured for
the 10 modified duplexes range between 0.050 and 0.003
(Table 4). Analogous to the quantum yields for the

4520 Nucleic Acids Research, 2011, Vol. 39, No. 10
DISCUSSION
0.34 which was recorded for AT in duplexes AA and TA in
viscous sucrose solutions. The difference compared to the
fundamental anisotropy of AT in a vitrified matrix (0.38)
was calculated to be due to an internal wobble of AT in the
DNA duplex of 16ꢀ. This value is higher than the
estimated wobble for the natural canonical bases (ꢂ5ꢀ),
but lower than the corresponding value for the
intercalating dye ethidium bromide (21ꢀ) (51).
Furthermore, melting experiments were performed to
examine the base pairing specificity of AT. Three of the
modified sequences (GA, CT, CA) were annealed with
strands containing an adenine, guanine or cytosine
opposite of AT instead of a thymine. Melting temperatures
recorded for AT-cytosine/guanine mismatches reveal an
average drop in melting temperatures of 16ꢀC, almost
twice as large compared to an AT-thymine match (8ꢀC).
The AT-cytosine/guanine mismatch melting temperatures
are in line with the average destabilization (14ꢀC) recorded
for a single-base mismatch of adenine–adenine in these
duplexes compared to their natural matching counter-
parts. It is therefore reasonable to assume that AT
exhibits some hydrogen-bonding with thymine but shows
virtually no base-pairing with guanine or cytosine.
Surprisingly, the same mismatch experiment performed
for an AT-adenine mismatch showed on average no
further destabilization of the duplexes (8ꢀC) compared
to the AT-thymine case. This suggests that AT is able to
form equally strong base pairs with thymine and adenine.
To the best of our knowledge, no similar findings have
been previously reported for other adenine analogues.
The putative AT.A base pair in Figure 6 would have a
similar overall shape to a Watson–Crick base pair and
would have good stacking interactions with surrounding
base pairs and, thus, constitutes a plausible structure.
Protonation of N(1) of adenine would provide a second
hydrogen bond. Additionally a possible third weakly
stabilizing C-H—N hydrogen bond to H(2) on adenine
may be formed (58). It should be mentioned that the
pKa of N(1) of adenine is approximately 4.0. However,
in double-stranded DNA, if it is or has the possibility of
being involved in H-bonding, it can be raised much higher.
Thus, the proposed base pair structure is presently specu-
lative and future high-resolution structural studies will be
In a recent study, we characterized a novel series of 8-
substituted adenosine analogues. That investigation
revealed promising features for triazole adenine (AT)
such as a high-quantum yield and a red-shifted absorption
band, allowing specific excitation outside the
DNA-absorption band (49). In the study presented here
we therefore decided to synthesize the 20-deoxyribose de-
rivative of AT in order to perform a comprehensive
photophysical and structural characterization when AT
is incorporated into DNA.
Structure and stability of DNA duplexes containing AT
To investigate the influence on B-DNA stability after in-
corporation of AT, we performed UV-melting experiments
on the 10 different 10-mer duplexes. These sequences
contain one AT flanked by varying combinations of
purines and pyrimidines as direct 30 and 50 neighbours.
The resulting melting data show a moderate destabiliza-
tion of duplexes containing AT by an average drop in
melting temperature of 8ꢀC compared to the natural
duplexes. The decrease in stability is dependent on the
bases surrounding AT. A more thorough investigation
shows that duplexes containing a pyrimidine 30 of AT
are the most stable. This is most likely due to a better
stacking between AT and the surrounding bases when it
is flanked by a pyrimidine as a 30 neighbour. The gen-
eral destabilization is most probably caused by steric
clashes of the substituent triazole ring and pentyl chain
in the 8-position of AT with the phosphate backbone
and sugar moieties. This steric clash would account for a
considerable energy cost if the base is to be accommodated
in the duplex in its anti conformation.
Similar observations have been made for 8-methoxy
and 8-bromo substituted adenine, which showed an
average destabilization of 6ꢀC in 11-mer DNA duplexes
(1.0 M NaCl, pH 7, 0.010 M phosphate, 0.001 M EDTA)
(54). It has also been shown previously that many
8-substituted purines show a preference for the syn con-
formation (55,56). However, calculations suggested a syn/
anti equilibrium to be present in DNA helices for other
analogues (57). The preferred orientation of the modified
base around the glycosidic bond in duplex DNA depends
on the hydrogen bonding preferences, which can change
depending on the bulky substituent and can improve the
relative stability of Hoogsteen pairs. Stabilization of the
syn orientation compared to the anti orientation helps to
explain the mutagenicity of bulky DNA adducts. In the
case of the AT(anti).T base pair the triazole moiety would
have strong unfavourable steric and electrostatic inter-
actions with the adjacent phosphodiester backbone,
whereas in the AT(syn) orientation there is the possibility
of a third weakly stabilizing C-H—O hydrogen bond to
thymine (58). In both versions of the AT.T base pair, the
pentyl group will project into the aqueous environment
and this could lead to entropic destabilization.
Despite this possible change in conformation, AT still
shows base pairing capacity to thymine and seems to be
stacked reasonably well in the DNA duplex in that case.
This can be concluded from a limiting anisotropy value of
Figure 6. Putative stabilized AT.A base pair. Only the sugar attached
to AT is shown and its triazole and pentyl chain are coloured grey. R1,
R2 and R3 represent the rest of the DNA structure.

Nucleic Acids Research, 2011, Vol. 39, No. 10 4521
necessary to accurately determine the base pairing
properties of AT.
minor differences in CD-signal can be detected where the
regular DNA bases absorb (60,63).
As was mentioned above, bulky DNA adducts can in-
fluence the hydrogen bonding preferences of the base, re-
sulting in potential mispairing involving the Hoogsteen
face. Deviations in the base pairing specificity have also
been found for other base analogues due to the introduced
modifications to the natural base. The adenine analogue
2-aminopurine is, for example, able to form base pairs
both with thymine and cytosine (28,59). Furthermore,
the base discriminating fluorescent analogue BPP can
form stable base pairs both with adenine and guanine (7).
The drop in melting temperature caused by AT (8ꢀC) is
not dramatic when compared to most other fluorescent
base analogues which have been incorporated into
DNA. For example, earlier observations for decamers
containing the fluorescent analogue 2-aminopurine have
shown a drop in stability of 10ꢀC (0.1 M KCl, 0.1 mM
EDTA, 21 mM DNA duplexes) (27). In another study
involving 14-mer duplexes a decrease of 6ꢀC in melting
temperature was observed after incorporation of
2-aminopurine in place of adenine (0.015 M sodium
citrate, pH 7.25, 50 mM DNA duplexes) (60). However,
in a recent study, a decrease in melting temperature of
merely 3.5ꢀC was recorded for 13-mer duplexes (10 mM
Na2HPO4/NaH2PO4, 150 mM NaCl, pH6.5, 11.9 mM
DNA duplexes) (61). Besides 2-aminopurine, other
adenine analogues such as 6MAP and DMAP on
average have shown a slight (2.4ꢀC) to moderate destabil-
ization (4.6ꢀC) in 21-mer duplexes (10 mM Tris, pH 7.5,
with 10 mM NaCl), respectively (16). Furthermore, the
pteridine guanine analogue 3-MI shows an average drop
Fluorescence properties of AT in single- and
double-stranded DNA
The emission maximum and the spectral envelope of AT
are virtually unaffected by incorporation into oligonucleo-
tides. On the other hand, as reported for virtually all base
analogues (5,15,16,24,26), incorporation of AT in oligo-
nucleotides causes a significant drop in fluorescence
quantum yield. Quantum yield values for AT in
single-stranded DNA (0.005–0.21) are on average 12
times lower than those recorded for the AT monomer in
water (0.61). These values are reduced approximately
another five times in duplexes (0.003–0.050).
Furthermore, the average lifetimes of AT in both single-
(0.9–1.5 ns; except GC: <ꢂ> = 2 ns) and double-stranded
DNA (0.2–1.2 ns) are also reduced compared to the
lifetime of the AT monomer in water (1.8 ns). With excep-
tion of the single-stranded samples AA and GC, showing a
mono-exponential decay, three lifetimes were recorded for
AT incorporated in all double- and single-stranded
samples. These changes in photophysical properties
when going from monomer AT to DNA-incorporated
AT may be explained by different levels of stacking and,
thus, electronic interaction with other bases in DNA. For
both the single- and double-stranded samples an increase
in the non-radiative rate constant (knr) as well as a
decrease in the radiative rate constant (kf) of AT can be
observed compared to its free monomeric form (data not
shown). First of all, the absorption of AT is most likely
decreased in DNA compared to the free monomer, as seen
for all natural bases, due to stacking interactions with the
surrounding bases. As a result the radiative rate constant,
kf, is lowered and gets influenced by the reduction of the
extinction coefficient since they are related through the
Strickler–Berg equation (64). This corresponds well with
the drop in quantum yield and decrease in kf (except for
strand AC) which we observed for the duplex samples
compared to the single strands. Second, several conform-
ations of AT might be present in DNA. Calculations
suggest that an increase in the dihedral angle between
the triazole ring and the adenine moiety (!), starting
in melting temperature (8.3ꢀC) comparable to
single-base mismatch under similar conditions (15).
a
Importantly, the CD-spectra recorded for the different
duplexes containing AT show a general signature for
regular B-form DNA. This means that the AT-modifica-
tion does not perturb the overall B-form of the DNA. Five
of the modified duplexes (CT, CA, TG, CC and AA) show
an almost identical CD-spectrum to their corresponding
natural duplexes. Surprisingly, no change in CD signal
was found for the duplexes AA and CT in the region
where only the AT absorption bands are situated.
Similar findings have been reported in the case of tCo
and currently we have no satisfactory explanation for
this phenomenon (23). Duplexes CA, TG and CC show
slight differences which correspond well to the AT- ab-
sorption bands. In contrast, the other duplexes show
more distinct differences compared to the CD-signature
of their corresponding natural duplexes (GA, GC, GG,
TA and AC). For the duplexes GA, GC and GG, the
CD is most perturbed, which may indicate that the
B-DNA structure is more distorted when AT is flanked
by a guanine at the 50-side. As expected, the deviations
in CD signals correspond well to the absorption difference
of AT and A in duplex DNA. Comparable results were
found for the CD spectra of duplexes containing the fluor-
escent analogue tC (62). Also the adenine analogue,
2-aminopurine, shows a particular significant CD-band
>300 nm when incorporated into duplex DNA, whereas
from a planar conformation (! = 0), results in
a
blueshifted absorption and decreased oscillator strength
of the lowest electronic transition (49) (B. Albinsson,
personal communication). It was previously suggested by
looking at the orbital diagrams of the model compound
9-methyl-8-(1H-1,2,3-triazol-4-yl)adenine that the single
bond character of the bond between the adenine and
triazole moiety of AT gains bond order in the excited
state, resulting in a planar excited state (49). However,
in DNA AT might be forced to remain in a twisted
geometry in the excited state which also results in
changes in oscillator strength compared to the monomer
AT, corresponding to different geometries of AT around
the dihedral bond. Again, since oscillator strength is pro-
portional to the radiative rate constant (kf) of the excited
state through the Strickler–Berg equation (64), these

4522 Nucleic Acids Research, 2011, Vol. 39, No. 10
different emitting species may also explain the multiple
fluorescence lifetimes observed. Finally, changes in
photophysical properties can also be explained by in-
creases in knr upon incorporation of AT in oligonucleo-
tides as a result of, for example, photoinduced electron
transfer from neighbouring bases, as will be further
described below.
CONCLUSION
We have performed a comprehensive photophysical and
structural characterization of the 20-deoxyribose derivative
of AT incorporated into DNA and in its monomeric form
and have found very promising properties of AT
compared to the widely used commercially available
2-aminopurine. AT causes only minimal structural per-
turbations to B-DNA. Furthermore, AT has a very
high-quantum yield as a monomer in water (0.61) as
well as in methanol (0.49). Inside DNA, the quantum
yield reaches values >20% in certain single strands and
5% in double strands, 10 to 5 times higher, respectively,
than corresponding values for 2-aminopurine. These
promising features should lead to future applications of
AT in studies concerning the structure, dynamics and
interactions of nucleic acids.
Not only we do notice a general decrease in quantum
yield upon incorporation of AT into DNA, but also sig-
nificant variations in fluorescence are observed when AT is
placed in various sequence contexts. This is a common
property of many fluorescent base analogues (15,16,65–
67). Sequence AA, with two adenines neighbouring AT,
shows the highest quantum yield value both for single-
(0.21) and double-stranded DNA (0.05). Quantum yield
values decrease for having an adenine (CA, TA, exception:
GA), a cytosine (GC, AC, CC) or a thymine (CT) flanking
AT on the 30-side. The lowest quantum yield values are
detected in the case where AT is flanked with two
guanines (GG; 0.005 ss; 0.003 ds) or with a guanine at
the 30-side (TG; 0.006 ss; 0.003 ds). This is not surprising,
since guanine has the lowest oxidation potential of all
natural bases (68) and therefore might transfer an
electron into the neighbouring AT resulting in a fast
relaxation to the ground state. This kind of quenching
pattern has been seen before both for the base
discriminating analogue BPP and 2-aminopurine when
flanked by a guanine (7,66). However, sequences that
have a guanine neighbouring AT at the 50-side (GC and
GA) show significantly higher quantum yields (0.042 and
0.018, respectively). This corresponds to previous obser-
vations made for the fluorescent base analogue tCo,
namely that both proximity to a guanine and structural
differences are important for quenching (23). These struc-
tural differences probably include parameters such as
relative orientation and stacking of AT and neighbouring
guanines.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
Joakim Karnbratt, Søren Preus and Dr Francois-
Alexandre Miannay are gratefully acknowledged for
their input concerning the photophysical properties of
AT and for technical assistance.
FUNDING
Swedish Research Council (to L.M.W.); Olle Engkvist
Byggmastare Foundation (to L.M.W. and M.G.);
European Community’s Seventh Framework Programme
entitled
READNA
(FP7/2007-2013;
grant
HEALTH-F4-2008-201418 to A.H.E.-S. and T.B.).
Funding for open access charge: Swedish Research
Council.
Finally, it is important to note that the maximum
quantum yield recorded for AT in single strands (0.21,
AA) is approximately 10 times higher than the maximum
value measured for the widely used dye 2-aminopurine.
Furthermore, the maximum quantum yield of AT when
incorporated into duplexes is approximately five times
higher than the corresponding value for 2-aminopurine.
It should also be noted that good lines of evidence
suggest that these values for 2-aminopurine are overesti-
mations since in some cases 2-aminopurine was
incorporated at the ends of the oligonucleotide (26).
Moreover, the maximum quantum yield values for AT in
single-stranded DNA reported here are also five and two
times higher, respectively, than for the pteridine adenine
analogues 6MAP (0.041) and DMAP (0.11) under similar
conditions (16). In duplexes, the fluorescence of 6MAP
and DMAP is quenched by an additional 2% compared
to the monomer fluorescence quantum yield. Furthermore
the brightness (ꢂꢁf ꢃ e) of AT in duplex DNA is approxi-
mately three times higher than for 2-aminopurine and es-
sentially the same as for 6MAP when comparing with
extinction in the lowest energy absorption band
(16,26,63).
Conflict of interest statement. None declared.
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