Getting DNA and RNA out of the dark with 2CNqA: A bright adenine analogue and interbase FRET donor

Article abstract of DOI:10.1093/nar/gkaa525

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase F?rster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.

Full text of DOI:10.1093/nar/gkaa525

Nucleic Acids Research, 2020
1
doi: 10.1093/nar/gkaa525
Getting DNA and RNA out of the dark with 2CNqA: a
bright adenine analogue and interbase FRET donor
2,3
Anna Wypijewska del Nogal 1, Anders F. Fu¨chtbauer 1,2, Mattias Bood
,
Jesper R. Nilsson 1, Moa S. Wranne 1, Sangamesh Sarangamath1, Pauline Pfeiffer1,
Vinoth Sundar Rajan1, Afaf H. El-Sagheer 5, Anders Dahle´n 6, Tom Brown
4
,
2
1,*
Morten Grøtli
and L. Marcus Wilhelmsson
1Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg SE-412 96,
Sweden, 2Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg SE-412 96,
Sweden, 3Medicinal Chemistry, Research and EarlyDevelopment, Cardiovascular, Renal and Metabolism (CVRM),
BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Pepparedsleden 1, Mo¨lndal, SE-431 83, Sweden, 4Chemistry
Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK, 5Chemistry Branch,
Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt and 6Discovery Sciences,
BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Pepparedsleden 1, Mo¨lndal, SE-431 83, Sweden
Received April 14, 2020; Revised May 15, 2020; Editorial Decision June 07, 2020; Accepted June 09, 2020
ABSTRACT
INTRODUCTION
While DNA stores the genetic information for a cell
to produce proteins, RNA is actively involved in the
(bio)chemistry and regulation of gene expression. In addi-
tion to early established mRNA, tRNA and rRNA, RNAs
like microRNA (miRNA), small interfering RNA (siRNA),
small nuclear RNA (snRNA), small nucleolar (snoRNA),
piwi-interacting RNA (piRNA), long non-coding RNA
(lncRNA), ribozymes and riboswitches have more recently
emerged as vital players in the regulation of protein syn-
thesis. These fundamental molecules of life have spurred
an increasing need to develop tools and techniques for
their study in multiple contexts, such as transcription, RNA
transport, the ribosomal machinery and mRNA trans-
lation, mRNA degradation, and several complex, RNA-
mediated mechanisms of gene silencing and epigenetic mod-
ification (1–3). Molecular biology and biochemistry assays,
supported by radioactive labelling and studies employing
external fluorophores (fluorophores attached covalently to
the 5ꢀ or 3ꢀ end of oligonucleotides; e.g. Cy-, Alexa- and
ATTO-dyes), were and still are vital techniques to provide
insights about nucleic acid function and related processes
(4–6). DNA is relatively static and structurally less versatile
than RNA. Hence, techniques like NMR, X-ray crystallog-
raphy and Cryo-EM have successfully provided significant
structure data for DNA (7,8). This is also true for certain,
structurally less dynamic RNAs (9–14). However, obtain-
ing structure and dynamics data (including information on
supertertiary structure, conformational ensembles and spa-
tiotemporal dynamics) for various forms of the structurally
With the central role of nucleic acids there is a need
for development of fluorophores that facilitate the
visualization of processes involving nucleic acids
without perturbing their natural properties and be-
haviour. Here, we incorporate a new analogue of ade-
nine, 2CNqA, into both DNA and RNA, and evaluate
its nucleobase-mimicking and internal fluorophore
capacities. We find that 2CNqA displays excellent
photophysical properties in both nucleic acids, is
highly specific for thymine/uracil, and maintains and
slightly stabilises the canonical conformations of
DNA and RNA duplexes. Moreover, the 2CNqA fluo-
rophore has a quantum yield in single-stranded and
duplex DNA ranging from 10% to 44% and 22% to
32%, respectively, and a slightly lower one (average
12%) inside duplex RNA. In combination with a com-
paratively strong molar absorptivity for this class of
compounds, the resulting brightness of 2CNqA in-
side double-stranded DNA is the highest reported
for a fluorescent base analogue. The high, relatively
sequence-independent quantum yield in duplexes
makes 2CNqA promising as a nucleic acid label and
as an interbase Fo¨rster resonance energy transfer
(FRET) donor. Finally, we report its excellent spectral
overlap with the interbase FRET acceptors qAnitro and
tCnitro, and demonstrate that these FRET pairs enable
conformation studies of DNA and RNA.
*To whom correspondence should be addressed. Tel: +46 317723051; Email: marcus.wilhelmsson@chalmers.se
ꢁ
C
The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
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2 Nucleic Acids Research, 2020
more pliable RNAs calls for novel and/or improved tech-
niques that can complement existing ones.
Fo¨rster resonance energy transfer (FRET) is
a
fluorescence-based technique that can provide both
structural and dynamics information, and in this respect
represents a complementary method to NMR and X-ray
crystallography to study DNA and, especially, RNA.
Such detailed structural and dynamics information has,
for example, been obtained using single-molecule FRET
combined with comprehensive analysis and modelling
(15). Unfortunately, the number of intrinsically fluorescent
entities in DNA and RNA that could be employed in
sensitive spectroscopy and microscopy techniques is scarce.
One exception is the mRNA cap - a naturally occurring
N7-modified guanosine that is present on the mRNA 5ꢀ
end. As a fluorophore, it has been successfully applied in
spectroscopic studies of mRNA-related processes, such
as protein synthesis (16) and mRNA turnover (17), as
well as in the characterisation of mRNA-binding proteins
(18–20). To overcome the shortage of intrinsic and min-
imally perturbing fluorophores in nucleic acids, we are
continuously expanding our library of fluorescent base
analogues (FBAs) (21–25). This class of fluorophores
(24) represents a versatile alternative to the commonly
used external fluorophores; they have the advantage of
being (i) non-bulky base-complementary mimics of natural
nucleobases suitable for incorporation into DNA/RNA,
preserving their canonical conformation and (ii) reporter
fluorophores that can be located close to or inside a ligand
(e.g. a small molecule, a protein, another nucleic acid
or a drug) binding site. A remaining challenge with the
utilization of fluorescent base analogues is their lower
brightness (εꢀF; molar absorptivity multiplied by the
fluorescence quantum yield) compared with conventional
external fluorophores such as those mentioned above.
However, recent studies show that the brightness gap
between internal and external fluorophores is decreasing
(26–28).
Figure 1. Structure of the studied 2CNqA, qAnitro and tCnitro nucleosides;
R1 is H for DNA and OH for RNA.
fore applicability as a nucleic acid label and as an interbase
FRET donor (30,33,34), we decided to investigate 2CNqA
in nucleic acids.
Herein, we characterise the photophysical properties of
2CNqA in both DNA and RNA and show that it can be
successfully used as a donor in FRET pairs with qAnitro (30)
and tCnitro (35) as acceptor nucleobases (Figure 1). We in-
vestigate the properties of 2CNqA in both B-form DNA
and A-form RNA, and its selectivity in base-pairing. We
find that our novel 2CNqA analogue is a highly promising
nucleic acid fluorophore for future utilisation in biophysi-
cal, biochemical and molecular biology contexts and that
it displays the highest average brightness inside DNA ever
reported for a fluorescent base analogue.
MATERIALS AND METHODS
Synthesis of the 2CNqA deoxyribonucleoside and ribonucle-
oside
The method developed for the preparation of the previ-
ously published qA derivatives, qAN1, qAnitro and pA, was
adopted for the synthesis of the 2CNqA-containing de-
oxyribonucleoside (28,30). The DMTr-protected phospho-
ramidite of 2CNqA (5) was synthesized over 13 steps with
an overall yield of 25% from commercially available 6-
chloro-7-iodo-7-deazapurine (1) (Scheme 1; for details, see
Supporting information (SI)).
The 2CNqA ribonucleoside was initially thought to be
accessible through the N-glycosylation of compound 2 with
a protected ribose sugar. However, this strategy turned out
to be incompatible with the majority of commercially avail-
able protected ribose derivatives. Instead, N-glycosylation
was performed as the first step, as in the original qA synthe-
sis protocol (29,36). Silyl–Hilbert–Johnson glycosylation of
deazapurine 1 with protected ribose 6 furnished compound
7 in moderate yield (37). Following our previously reported
protocol, Miyaura-borylation of 7 provided pinacol ester 8
in a good yield (30,38). 3-Amino-4-iodobenzonitrile (9) was
reacted with di-tert-butyl dicarbonate to yield 10, which was
used in a Suzuki-Miyaura cross-coupling with 4 to produce
compound 11. Intramolecular cyclisation was achieved us-
ing DABCO and DBU to furnish compound 12 after a
global deprotection using sodium methoxide (29). The final
phosphoramidite building block for solid-phase synthesis
In particular, we demonstrated that the brightness of our
parent adenine analogue, quadracyclic adenine (qA) (29)
can be significantly improved by replacing its outer ring
with a pyridine or a naphthalene, resulting in fluorophores
of a brightness that is excellent for a fluorescent base ana-
logue in aqueous solution, qAN1 (εꢀF = 1700 M−1 cm−1
)
and pA (εꢀF = 10100 M−1 cm−1), respectively (28,30). Un-
fortunately, when incorporated into DNA, the fluorescence
quantum yields and lifetimes of the two adenine derivatives
decrease and are sensitive to the neighbouring bases (28,30).
The brightness of pA inside DNA is still respectable (on
average 1400 M−1 cm−1) and close to the highest average
brightness inside DNA (2000 M−1 cm−1, as reported for
tCO) (31), but compared with commonly used external fluo-
rophores, which in general have brightness values well above
104 M−1 cm−1, there is still room for improvement. In our
recent study on outer ring substituents of qA, the 2CNqA
nucleobase (Figure 1), bearing a cyano (CN) group in the 2-
position of the outer ring, was found to possess a high and
stable quantum yield in a variety of solvents (32). Therefore,
in our search for an adenine analogue which, like tCO (31),
would exhibit a high and stable quantum yield regardless
of sequence context, rendering a high brightness and there-

Nucleic Acids Research, 2020 3
Scheme 1. Synthesis of the 2CNqA-DNA (5) and 2CNqA-RNA (13) phosphoramidite building blocks: (a) NaH, MeCN, 0◦C, 30 min; (b) 1.2 eq 3, 0◦C, 5
min, then RT, 30 min, 72% over two steps; (c) Bis(trimethylsilyl)acetamide, TMSOTf, MeCN, 80◦C, 1 h, 60%; (d) 2 mol% Pd(PPh3)4, HBPin, Et3N, THF,
80◦C, 36 h, 76%; (e) Boc2O, NaHMDS, THF, −78◦C, 1 h, 81%; (f) 1 eq 10, 5 mol% PdCl2(PPh3)2, K2CO3, DME, 80◦C, 55 h, 83%; (g) DBU, DABCO,
DMF, 70◦C, 12 h; (h) NaOMe, MeOH, RT, 1 h, 46% over two steps; (i) DMTrCl, Pyridine, RT, 3 h, 80%; (j) TBDMSCl, AgNO3, THF, Pyridine, RT, 7 h,
67%; (k) CEPCl, DIPEA, THF, RT, 20 h, 96%.
was prepared using standard protection and phosphoryla-
tion chemistry to yield compound 13 over a total of eight
steps and an overall yield of 9% from 1.
account for the base-stacking interactions. The following
molar absorptivities at 260 nm were used: εA = 15300 M−1
cm−1, εC = 7400 M−1 cm−1, εG = 11800 M−1 cm−1, εT =
9300 M−1 cm−1 (39), ε2CNqA = 14600 M−1 cm−1 (32) and
Synthesis of modified and unmodified DNA and RNA
oligonucleotides
εqAnitro = 12900 M−1 cm−1 (30). For RNA oligonucleotides,
molar absorptivities at 260 nm were calculated using the
Integrated DNA Technologies (IDT) OligoAnalyzer (http:
//eu.idtdna.com), with the modified base replaced by its un-
modified counterpart, and correcting for the difference in
molar absorptivity at 260 nm between unmodified base and
The sequences of 2CNqA-containing DNA and RNA
oligonucleotides used in this study are presented in Tables
1–3. See the SI for synthesis and characterisation details.
Complementary oligonucleotides to these sequences (un-
modified and modified, which either contain qAnitro (DNA)
or tCnitro (RNA)), as well as the mismatched oligonu-
cleotides of CT, GA and TA DNA sequences (Table 1) with
thymine in position 5, substituted by A, C or G, were ei-
ther synthesised (see the SI for details) or purchased from
ATDBio Ltd.
2CNqA (ε2CNqA = 14600 M−1 cm−1) or tCnitro (εtCnitro
=
9700 M−1 cm−1) (33). Using the Beer−Lambert law, the
molar absorptivity of the 2CNqA ribonucleoside at 260 nm
and the lowest-energy absorption maximum (λAbs,Max) =
356 nm was determined by measuring the absorption of
its aqueous solution (MQ water, Millipore) at three differ-
ent concentrations. Experiments on DNA were performed
in 13 mM sodium phosphate buffer, containing 100 mM
General information on photophysical measurements
NaCl, pH 7.50
0.05 and on RNA in 10 mM sodium
phosphate buffer, containing 100 mM NaCl and 1.0 mM
EDTA, pH 7.40 0.05. All measurements were conducted
at room temperature (RT, ca 22◦C), unless stated otherwise.
To form duplexes, a 2CNqA-modified strand was mixed
with 15% excess of its complementary strand. Prior to mea-
surements, DNA duplex samples were heated to 95◦C for
10 min, incubated at 95◦C for 5 min and cooled to 5◦C at a
All photophysical measurements were carried out in 0.3 or
0.4 cm pathlength quartz cuvettes (Hellma). The concen-
tration of both single- and double-stranded DNA/RNA
oligonucleotides was 4.0 ␮M, unless stated otherwise.
The molar absorptivities of DNA oligonucleotides were
approximated by the linear combination of molar absorp-
tivities of individual nucleotides and multiplied by 0.9 to

4 Nucleic Acids Research, 2020
Table 1. Sequences of 10 nt 2CNqA-ssDNAs and melting temperature (Tm) of their duplexes and their unmodified duplex counterpartsa
Sequence namea
5ꢀ-3ꢀ Sequence (X = 2CNqA)
2CNqA-dsDNA Tm [◦C]
Unmodified dsDNA Tm [◦C]
ΔTm [◦C]
AA
AC
AG
AT
CA
CC
CG
CT
GA
GC
GG
GT
TA
TC
TG
TT
CGC AAX ATC G
CGC AAX CTC G
CGC AAX GTC G
CGC AAX TTC G
CGC ACX ATC G
CGC ACX CTC G
CGC ACX GTC G
CGC ACX TTC G
CGC AGX ATC G
CGC AGX CTC G
CGC AGX GTC G
CGC AGX TTC G
CGC ATX ATC G
CGC ATX CTC G
CGC ATX GTC G
CGC ATX TTC G
42.4
48.8
45.5
46.1
52.1
58.1
55.8
56.0
45.0
51.6
0.3
0.3
0.2
0.1
0.2
0.3
0.1
0.3
0.1
0.3
43.3
47.0
45.8
43.5
46.9
50.5
49.5
47.0
45.3
49.0
48.0
45.6
41.1
43.5
43.5
40.5
− 0.9
1.8
− 0.3
2.6
5.2
7.6
6.3
9.0
− 0.3
2.6
49.1b
48.7b
44.4
49.5
47.4b
47.4
1.1
3.1
3.3
6.0
3.9
6.9
0.3
0.3
0.4
aSequence names indicate bases surrounding 2CNqA on the 5ꢀ and 3ꢀ side. Measured in 13 mM sodium phosphate buffer, 100 mM NaCl, pH 7.5.
b
Measured only once.
Table 2. Sequences of 33 nt 2CNqA-ssDNAs and complementary qAnitro-ssDNAs used in the DNA FRET study
Sequence namea
5ꢀ-3ꢀ Sequence (X = 2CNqA, Y = qAnitro
)
DNA-D0
DNA-D7
DNA-D9
DNA-D11
DNA-A0
DNA-A13
DNA-A14
DNA-A19
DNA-A20
CGA TCA AAA AAA ATT ACG ATT ATA AGG AGG AGG
CGA TCA XAA AAA ATT ACG ATT ATA AGG AGG AGG
CGA TCA AAX AAA ATT ACG ATT ATA AGG AGG AGG
CGA TCA AAA AXA ATT ACG ATT ATA AGG AGG AGG
CCT CCT CCT TAT AAT CGT AAT TTT TTT TGA TCG
CCT CCT CCT TAT YAT CGT AAT TTT TTT TGA TCG
CCT CCT CCT TAT AYT CGT AAT TTT TTT TGA TCG
CCT CCT CCT TAT AAT CGT YAT TTT TTT TGA TCG
CCT CCT CCT TAT AAT CGT AYT TTT TTT TGA TCG
aSequence names indicate position of the donor (D) or the acceptor (A) in the oligonucleotide counted from its 5ꢀ end, whereas DNA-D0 and DNA-A0
denote unmodified reference sequences.
Table 3. Sequences of 25 nt 2CNqA-ssRNAs and complementary tCnitro
ssRNAs used in the RNA FRET study
-
Absorption and absorption-based nucleic acid melting curves
Absorption spectra were recorded from 230 to 600 nm on
Varian Cary spectrophotometer (model 4000 or 5000) us-
ing a spectral bandwidth (SBW) of 1.0 nm. The data in-
terval was 0.5 nm, and the signal averaging time was 0.1 s.
The absorption (UV) DNA and RNA melting curves were
measured using the same instrument equipped with a multi-
cell temperature block by monitoring the absorption at 260
nm versus temperature. Both the heating and the cooling
curves over the range 20–90◦C were measured at 1.0◦C in-
tervals. The temperature ramp was 1.0◦C/min. The duplex
concentration was 2.0 ␮M in all melting experiments. The
melting temperature (Tm) was determined as the maximum
of the first derivative of the melting curve after FFT filter
smoothing.
Sequence
namea
5ꢀ-3ꢀ Sequence (X = 2CNqA, Y = tCnitro
)
RNA-D0
RNA-D6
RNA-D7
RNA-D12
RNA-D13
RNA-A0
RNA-A6
RNA-A8
RNA-A10
CGA CAA AAU CAA AAU GCG UGA UUG G
CGA CAX AAU CAA AAU GCG UGA UUG G
CGA CAA XAU CAA AAU GCG UGA UUG G
CGA CAA AAU CAX AAU GCG UGA UUG G
CGA CAA AAU CAA XAU GCG UGA UUG G
CCA AUC ACG CAU UUU GAU UUU GUC G
CCA AUY ACG CAU UUU GAU UUU GUC G
CCA AUC AYG CAU UUU GAU UUU GUC G
CCA AUC ACG YAU UUU GAU UUU GUC G
aSequence names indicate position of the donor (D) or the acceptor (A) in
the oligonucleotide counted from its 5ꢀ end, whereas RNA-D0 and RNA-
A0 denote unmodified reference sequences.
Circular dichroism (CD)
rate of 0.13◦C/min. To hybridize RNA duplexes, the sam-
ples were heated to 85◦C over 15 min, incubated at 85◦C
for 15 min and cooled to 5◦C at a rate of 1◦C/min. In the
FRET and quantum yield measurements, a 30% excess of
acceptor strand was used to ensure complete hybridisation,
i.e. that every donor strand molecule is bound to an accep-
tor strand. Data were obtained from two or more measure-
ments and are presented as the arithmetic mean standard
error of the mean (SEM), unless stated otherwise.
CD spectra were recorded from 200 to 450 nm for the 10 bp
2CNqA-DNAs (Table 1) and from 200 to 500 nm for the 25
bp 2CNqA-RNAs (Table 3) on a Chirascan CD spectrome-
ter (Applied Photophysics), using an SBW of 1.0 nm. Three
subsequent scans were collected with 1.0 nm intervals and
0.5 s integration time and then averaged and background
subtracted. Samples were measured at a concentration of
2.0 ␮M.

Nucleic Acids Research, 2020 5
Steady-state fluorescence
where ꢀF is the fluorescence quantum yield and the non-
radiative decay rate constant, kNR is given by:
Steady-state fluorescence measurements were carried out
using a Spex Fluorolog 3 spectrofluorometer (JY Horiba).
Emission spectra were recorded from 365 to 650 nm, with
the excitation at 355 nm for DNAs and at 354 nm for RNAs.
The slit widths for excitation and emission were set at 1.6
nm. The data interval was 1.0 nm and the integration time
was 0.1 s. The FRET efficiency in steady-state experiments
(EFRET,SS) was determined from the equation:
kR
ꢀF
kNR
=
− kR
For details regarding the determination of the fluores-
cence quantum yield (ꢀF), the spectral overlap integral
(JDA) and theoretical FRET efficiency (EFRET,The), see the
Supplementary methods.
FDA
EFRET,SS = 1 −
FD
Photodegradation quantum yield determination
where FDA is the integrated fluorescence emission intensity
of the donor in the presence of the acceptor and FD is the in-
tegrated fluorescence emission intensity of the donor alone.
The photodegradation quantum yield of the 2CNqA donor
in the absence of the acceptor (ꢀPd,D) for the single- and
double-stranded DNAs and RNAs (containing 2CNqA
flanked by two adenines) was determined by measuring
the rate of fluorescence decay (2CNqA bleaching) and
comparing it to the ring closing reaction of DAE6 (a di-
arylethene photoswitch employed as an actinometric ref-
Time-resolved fluorescence
Time-resolved fluorescence decays were measured using a
time-correlated single-photon counting (TCSPC) fluores-
cence lifetime spectrometer (LifeSpec II, Edinburgh Ana-
lytical Instruments), equipped with a 377 nm pulsed diode
laser (PicoQuant model LDH-P-C-375) as the excitation
source (FWHM pulse width was 1.0 nm and 70 ps with re-
spect to wavelength and time, respectively), and a R3809U-
50 microchannel plate photomultiplier (Hamamatsu). The
emission monochromator was set to 446 nm. The data were
collected using a pulse frequency of 5.0 MHz and 200 ns
time range until 10 000 counts were recorded in the top
channel out of 2048 channels used. The instrument response
function (IRF) was measured at the same settings of the ap-
paratus. Average fluorescence lifetimes (<τ>) were deter-
mined by fitting bi- or triexponential functions with recon-
volution to the decay curves and calculating the amplitude-
erence; Supplementary Figure S9) in 1,4-dioxane (ꢀPd,R
=
0.51 (41)) using the same irradiation conditions and sam-
ple volume. DAE6 can be isomerized photonically from its
non-fluorescent open-ring isomer to the fluorescent closed-
ring isomer using irradiation in the UV region. The ring
closing reaction of DAE6 and the 2CNqA bleaching were
both performed in the Spex Fluorolog 3 spectrofluorometer
(JY Horiba) using λExc = 365 nm (excitation slit at 7.0 nm)
while observing the emission (emission slit at 0.5 nm) at 445
nm for 2CNqA and 580 nm for DAE6. The excitation slit
was selected to let through a considerable amount of exci-
tation light in order to capture the photodegradation event
on the hour time-scale. Continuous irradiation and sam-
ple stirring were applied and the post-irradiation isomeric
state of DAE6 was confirmed by absorption spectroscopy
(Supplementary Figure S9). To determine ꢀPd,D, a mono-
exponential function was fitted to the fluorescence intensity
decay of the sample (and fluorescence intensity growth of
the reference), yielding the time constant, τ:
weighted averages:
ꢀ
i αi τi
ꢀ
ꢂτꢃ =
i αi
where τi is the fluorescence lifetime of the ith component
and αi is the amplitude of the ith component. The fit-
ting was performed using FluoFit Pro v.4 software (Pico-
Quant). The FRET efficiency in the time-resolved experi-
ments (EFRET,TR) was determined from the equation:
y = y0 + A1e−(x−x )/τ
0
ꢀPd,D was then obtained from the equation:
ε(λExc)RτR
ꢀPd,D = ꢀPd,R
ꢂτDA
ꢃ
ε(λExc)SτS
EFRET,TR = 1 −
ꢂτDꢃ
where ε(λExc) is the molar absorptivity at the excitation
wavelength, and subscripts S and R refer to the sample and
reference, respectively.
where <τDA> is the average fluorescence lifetime of the
donor in the presence of the acceptor and <τD> is the aver-
age fluorescence lifetime of the donor in the absence of the
acceptor.
The final experimental FRET efficiency (EFRET,Exp
)
Orientation of the donor and acceptor transition dipoles
was calculated as the weighted average of EFRET,SS and
EFRET,TR, with the weights given by the reciprocal squares
of the corresponding SEMs. The error in EFRET,Exp is the
higher of the internal and external weighted variances (40;
NIST/SEMATECH e-Handbook of Statistical Methods,
http://www.itl.nist.gov/div898/handbook/).
Time-dependent density functional theory (TDDFT)
methodology was used as described previously (32) to com-
putationally predict the orientation of the lowest energy
transition dipole moment of 2CNqA and qAnitro. These
values and the experimentally determined orientation for
the transition dipole moment of the tCnitro acceptor were
used together with nucleic acid B- and A-form, respectively,
to calculate the phase angle.
The radiative decay rate constant, kR is given by:
ꢀF
kR =
ꢂτꢃ

6 Nucleic Acids Research, 2020
Figure 2. Left: Representative circular dichroism spectra of a 10 bp 2CNqA-dsDNA (AA; black line) and its unmodified counterpart (blue line). Mea-
sured in 13 mM sodium phosphate buffer, 100 mM NaCl, pH 7.5 at RT. Right: Circular dichroism spectra of 25 bp dsRNAs used in the FRET study:
2CNqA-dsRNA (RNA-D13:RNA-A0; red line) and the corresponding donor-acceptor duplex, 2CNqA-tCnitro-dsRNA (RNA-D13:RNA-A10; blue line),
acceptor-only duplex, tCnitro-dsRNA (RNA-D0:RNA-A10; navy line) and unmodified dsRNA (RNA-D0:RNA-A0; black line). Measured in 10 mM
sodium phosphate buffer, 100 mM NaCl, 1.0 mM EDTA, pH 7.4 at RT.
RESULTS AND DISCUSSION
which are general to all duplexes investigated (Supplemen-
tary Figure S2), can be ascribed to differences in molar ab-
sorptivity of the adenine that is exchanged with a 2CNqA
in the modified duplex. As our previously studied fluores-
cent adenine analogues (qA (29), qAN1 (30) or pA (28)),
no CD band outside the nucleic acid wavelength range and
corresponding to the long-wavelength absorption band of
2CNqA can be observed (28–31).
Design of the 2CNqA-containing DNA and RNA oligonu-
cleotides
To investigate the photophysical and physicochemical prop-
erties of 2CNqA as an emissive adenosine mimic inside
nucleic acids, 16 DNA sequences were synthesised, bear-
ing 2CNqA at the same position within a 10-mer oligonu-
cleotide, but flanked with all possible combinations of
neighbouring canonical bases (Table 1). To evaluate the po-
tential of 2CNqA as a FRET donor, two previously devel-
oped acceptor entities were used, qAnitro (30) and tCnitro
(33). One of the FRET pairs, 2CNqA-qAnitro, was incor-
porated into DNA oligonucleotides (33 nt; Table 2) and the
second one, 2CNqA-tCnitro into RNA oligonucleotides (25
nt; Table 3), to examine this new FRET donor in both types
of nucleic acids. The RNA sequences of the FRET investi-
gation were also used in a less comprehensive study (com-
pared to the one of DNA described above) of the structure,
base analogue and photophysical properties of 2CNqA in-
side RNA.
The UV-melting experiments also clearly suggest that the
B-form secondary structure of dsDNA is preserved upon
2CNqA-incorporation. The melting curves of the 16 DNA
duplexes containing the 2CNqA have overall the same
shape as the curves of their unmodified counterparts (data
not shown). The melting temperature (Tm) of unmodified
and corresponding 2CNqA-dsDNAs are summarised in Ta-
ble 1. On average, 2CNqA incorporation increases the sta-
bility of DNA duplexes by 3.6◦C and ranges from a minor
destabilising effect (less than -1.0◦C) to a stabilising effect of
up to 9.0◦C. It is noteworthy that DNA duplexes containing
2CNqA demonstrate higher average thermal stability than
the ones previously reported for FBAs, pA (ꢁTm = 1.1◦C
(28)), qAN1 (ꢁTm = 2.9◦C (30)) and qA (ꢁTm = 3.0◦C
(29)). In a similar manner to qAN1, a higher stabilization
impact on dsDNAs is observed when 2CNqA has pyrim-
idines as nearest neighbours. For AA, AG and GA as near-
est neighbours, a small decrease in Tm is observed compared
to unmodified duplexes: −0.9◦C, −0.3◦C and −0.3◦C, re-
spectively. An analogous decrease in melting temperature
has previously been observed for qA with AA neighbours
(29), for qAN1 with AA and GA neighbours (30) and for
pA with AA, AG and GA neighbours (28).
Conformation, stability and base-pairing selectivity of
2CNqA-containing DNA- and RNA-duplexes
To study the impact of 2CNqA on the conformation of
nucleic acids, CD spectra were recorded for the 2CNqA-
dsDNAs (Figure 2, left; Supplementary Figures S1 and S2)
and 2CNqA-dsRNAs (Figure 2, right) in comparison to
their unmodified counterparts. As for unmodified DNA du-
plexes, the CD spectra of the 2CNqA-containing DNA du-
plexes are characterised by a positive band at about 260–280
nm and a negative band around 245 nm (Figure 2, left; Sup-
plementary Figures S1 and S2). This indicates that 2CNqA-
dsDNAs adopt the canonical double helix conformation, B-
DNA. The deviations from the CD of unmodified duplexes,
Importantly, we also find 2CNqA to be highly specific for
base-pairing with thymine (T) in DNA duplexes (Figure 3
and Supplementary Table S1). If the thymine of the com-
plementary strand opposite the 2CNqA is replaced by ade-
nine (A), cytosine (C) or guanine (G), the Tm of the duplex

Nucleic Acids Research, 2020 7
Table 4. The melting temperature (Tm) of a 25 bp unmodified se-
quence (RNA-D0:RNA-A0) and the corresponding 2CNqA-dsRNA
(RNA-D13:RNA-A0), tCnitro-dsRNA (RNA-D0:RNA-A10) and doubly-
labelled dsRNA (RNA-D13:RNA-A10)a
dsRNA
Tm [◦C]
ꢁTm [◦C]
RNA-D0:RNA-A0
RNA-D13:RNA-A10
RNA-D13:RNA-A0
RNA-D0:RNA-A10
67.0 0.1
68.7 0.2
68.5 0.2
68.2 0.3
–
1.7
1.5
1.2
aMeasured in 13 mM sodium phosphate buffer, 100 mM NaCl, 1.0 mM
EDTA, pH 7.4.
ficity of adenine for thymine, and slightly stabilizes the
canonical B-form helix structure of DNA. The effect of a
2CNqA modification in RNA is similar to that in DNA: the
canonical A-form conformation of RNA is being adopted
and a slight increase in duplex stability is observed.
Photophysical properties of 2CNqA in single- and double-
stranded DNA
Figure 3. The difference in melting temperature (ꢁTm) between three 10
bp 2CNqA-dsDNA (CT, GA and TA) containing the complementary
2CNqA-T pair and the corresponding duplexes containing the 2CNqA-A,
2CNqA-C or 2CNqA-G mismatches. Measured in 13 mM sodium phos-
phate buffer, 100 mM NaCl, pH 7.5.
2CNqA has a high fluorescence quantum yield as a
monomer in aqueous solution (42%) as well as a high
brightness value (εꢀF = 4500 M−1 cm−1) (32). Here, we in-
vestigate its fluorophore properties inside DNA (Table 1)
and will return in the RNA FRET study below to a smaller
photophysical characterisation of 2CNqA in RNA employ-
ing the RNA FRET sequences (Table 3). To understand
how various neighbouring bases inside DNA influence the
photophysical properties of 2CNqA, we investigated all 16
possible base surroundings (see Table 1 for sequences and
sequence names). A detailed summary of the photophysi-
cal properties of 2CNqA incorporated into the 16 ssDNA
sequences as well as their duplex counterparts can be found
in Table 5.
The 2CNqA-DNA monomer has its lowest-energy ab-
sorption (λAbs,Max) and emission maxima (λEm,Max) at
356 and 480 nm, respectively (32). The corresponding ab-
sorption maxima of 2CNqA in ssDNA and dsDNA are
found at virtually the same positions as for the monomer
and are centered at approximately 358 and 356 nm, respec-
tively, across the different combinations of neighbouring
bases (Figure 4, left, Table 5 and Supplementary Figures S4
and S5). Since the natural nucleobases do not absorb above
300 nm (42), this important feature of 2CNqA allows for its
selective excitation inside DNA.
The emission peak of 2CNqA in DNA is blue-shifted
compared to the monomer and positioned between 452 nm
and 457 nm in ssDNA and 448 nm and 459 nm in dsDNA
(Figure 4, right, Table 5 and Supplementary Figures S6 and
S7). The observed blue-shift of 2CNqA inside DNA is most
likely an effect of the less polar environment, which leads to
a reduced solvent relaxation - an effect commonly observed
for fluorescent base analogues inside nucleic acids (4).
When incorporated into ssDNA, 2CNqA displays a high
fluorescence quantum yield, ꢀF, ranging from 10% for
double-T neighbours to 44% for double-A neighbours (Ta-
ble 5). The lowest ꢀF values were obtained for sequences
containing thymine as the nearest neighbour (TT, TC, TG,
TA, CT, GT and AT; 10–18%), whereas the highest ones
significantly decreases (by −10.5 to −19.6◦C; Figure 3 and
Supplementary Table S1). Regardless of the nearest neigh-
bours to 2CNqA, pyrimidines (CT), purines (GA) or a com-
bination of the two (TA), the highest destabilization effect is
observed for the 2CNqA-A mismatch (−15.2 to −19.6◦C).
We next investigated the behaviour of 2CNqA as an ade-
nine analogue in RNA duplexes. As can be seen in Figure
2, right, the unmodified and modified (2CNqA, tCnitro or
both) RNA duplexes display a positive band near 265 nm
and a negative band at 210 nm. Such spectral features in
the CD of nucleic acids correspond to the canonical A-form
RNA. As for DNA, the long-wavelength absorption band
of 2CNqA in RNA is not observed in the CD (Figure 2,
right, RNA-D13:RNA-A0). The same is true for the FRET
acceptor, tCnitro (Figure 2, right, RNA-D0:RNA-A10). The
shape of the UV-melting curves of the 2CNqA-containing
and doubly modified RNA duplexes corresponds well to the
profile of their unmodified counterpart, RNA-D0:RNA-
A0 (Supplementary Figure S3), although slightly shifted to-
wards higher temperatures (Table 4). This gives further sup-
port that the A-form RNA is formed when an adenine is re-
placed by 2CNqA and a cytosine is replaced by tCnitro. The
Tm of the RNA-D13:RNA-A0 2CNqA-modified duplex is
1.5◦C higher than that of the unmodified RNA-D0:RNA-
A0, indicating that 2CNqA incorporation into RNA duplex
in place of an adenine slightly increases RNA stability and
that 2CNqA serves well as an adenine analogue also inside
RNA. The small effect of 2CNqA positioned between AA
neighbours on Tm is in line with the small effect found on
Tm for 2CNqA between AA neighbours in DNA duplexes
(Table 1).
In summary, based on the results of the conformation,
stability and base-pairing study of 2CNqA in DNA and
RNA, we conclude that 2CNqA is an excellent mimic of
natural adenine in both nucleic acids. It preserves the speci-

8 Nucleic Acids Research, 2020
Table 5. Photophysical properties of the sixteen 10 nt 2CNqA-ssDNAs and -dsDNAsa
ssDNA
dsDNA
λAbs
NN [nm]
λEm
ε
ꢀF
[%]
εꢀF
[M−1 cm−1
<τ>
kR
kNR
λAbs
λEm
ε
ꢀF
[%]
εꢀF
[M−1 cm−1
<τ>
kR
kNR
[nm] [M−1 cm−1
]
]
[ns]
[107 s−1
]
[108 s−1
]
[nm] [nm] [M−1 cm−1
]
]
[ns]
[107 s−1
]
[108 s−1
]
AA
AC
AG
AT
CA
CC
CG
CT
GA
GC
GG
GT
TA
TC
TG
TT
357
358
358
357
358
358
359
357
358
359
358
357
358
359
360
358
452
453
453
453
457
454
453
456
453
454
453
454
456
455
453
456
10300
10400
10200
10600
9700
9500
9400
9700
9700
10100
9500
10000
9800
42
35
35
18
33
27
25
15
40
28
40
17
15
13
14
10
4300
3600
3500
1900
3200
2600
2400
1500
3900
2800
3800
1700
1400
1300
1300
1000
11
3.8
3.7
4.0
3.8
3.9
3.8
4.1
3.8
3.8
3.8
4.0
4.0
4.0
3.9
4.2
3.6
0.53
0.69
0.76
1.7
0.78
1.0
1.2
2.1
0.56
0.97
0.61
2.0
355
355
356
355
356
356
356
357
355
355
357
356
356
357
358
357
453
451
454
449
458
455
459
451
455
451
456
448
456
456
459
449
9800
10100
9700
10000
8800
8700
8500
9000
9400
9500
9000
9700
9400
8800
8200
9200
31
29
32
32
26
28
27
31
26
25
29
29
22
26
27
29
3000
2900
3100
3200
2300
2400
2300
2700
2400
2400
2700
2800
2100
2300
2200
2700
9.5
9.2
9.7
9.6
8.5
9.1
9.2
9.7
8.5
8.3
9.2
9.4
7.4
8.8
8.9
9.3
3.3
3.2
3.3
3.4
3.0
3.0
3.0
3.2
3.0
3.0
3.2
3.1
3.0
3.0
3.0
3.1
0.73
0.77
0.70
0.71
0.87
0.79
0.79
0.72
0.87
0.90
0.77
0.76
1.0
9.4
8.6
4.7
8.6
7.1
6.2
4.0
11
7.4
9.9
4.2
3.7
3.4
3.3
2.8
2.3
2.5
2.7
3.2
9600
9600
10000
0.83
0.82
0.76
aλAbs: absorption maximum, λEm: emission maximum, ε: molar absorptivity, ꢀF: fluorescence quantum yield, εꢀF: brightness, <τ>: average fluorescence lifetime, kR: radiative
decay rate constant, kNR: non-radiative decay rate constant; Measured in 13 mM sodium phosphate buffer, 100 mM NaCl, pH 7.5 at RT. The ꢀF was determined at λExc = 355
nm with quinine sulfate in 0.5 M H2SO4 (ꢀF,R = 0.55) as the reference. The <τ> was determined at λExc = 377 nm and λEm = 446 nm. The reported ε, ꢀF and <τ> values are
the arithmetic mean of two or more measurements. Their standard errors of the mean (SEM) can be found in Supplementary Table S2. The reported kR and kNR values were
calculated as described in Materials and Methods.
Figure 4. Absorption (left) and emission (right; λ
= 355 nm) spectra of a representative 10 nt 2CNqA-ssDNA with adenines as neighbours (AA) of
Exc
2CNqA (blue) and its duplex counterpart (black). Measured in 13 mM sodium phosphate buffer, 100 mM NaCl, pH 7.5 at RT.
were found for sequences containing two purines as near-
est neighbours (AG, GG, GA and AA; 36–44%). These lat-
ter, high quantum yields of 2CNqA in ssDNA are compa-
rable to the quantum yield of 2CNqA alone (42% in water
(32)), suggesting a minimal quenching in these sequences
relative to aqueous surroundings. Similar quenching prop-
erties (strong for neighbouring thymine and minimal for
neighbouring purines) have been observed for our recently
reported quadracyclic adenine analogue, qAN1 (30).
Remarkably, the quantum yield of 2CNqA shows signif-
icantly less variation between different sequences when in-
corporated into dsDNA (ꢀF ranges from 22 to 32%; Ta-
ble 5). Such low dependence of sequence surroundings on
the quantum yield of a fluorescent base analogue has pre-
viously only been reported for the tricyclic cytosine ana-
logues tC (43) and tCO (31). As was observed for qAN1
(30), the quenching effect of neighbouring thymine is de-
creased when going from single- to double-stranded DNA.
Moreover, the difference in quenching between purines and
pyrimidines is not as pronounced for dsDNAs as for ssD-
NAs (Table 5). On average, the quantum yield of 2CNqA
in dsDNA is 28.1%, which is comparable to that in single
strands (26.1%) and 1.5-fold lower compared to the 2CNqA
monomer (42% (32)). The reported quantum yields inside
DNA are high in comparison to those of most reported flu-
orescent base analogues and together with molar absorp-
tivities of, on average, 9900 M−1 cm−1 in ssDNA and 9200
M−1 cm−1 in dsDNA (Table 5), the average brightness of
2CNqA reaches 2500 M−1 cm−1 and 2600 M−1 cm−1 for
single- and double-stranded DNA, respectively. These val-
ues are the highest single-photon excitation brightness val-
ues reported so far for a fluorescent base analogue in nu-
cleic acid systems (for a comparative table, see the report
by Bood et al. (28)); e.g. average brightness in dsDNA is
510 M−1 cm−1 for qAN1, 760 M−1 cm−1 for tC, 1400 M−1
cm−1 for pA, 1700 M−1 cm−1 for 6-MI and 2000 M−1 cm−1

Nucleic Acids Research, 2020 9
for tCO). Moreover, unlike previously reported A analogues
such as our bright qAN1 and pA, 2CNqA has a high quan-
tum yield and is bright regardless of sequence context in
duplex DNA. These features are important for future ap-
plications of 2CNqA as an internal nucleic label and as a
donor in interbase FRET (vide infra).
corresponding duplexes, the RNA (ꢀPd,D = 0.17 0.02%)
is slightly more resistant to photodegradation than the
DNA (ꢀPd,D = 0.25 0.01%). The ꢀPd,D values also high-
light that, regardless of the nucleic acid type, photobleach-
ing is significantly less prominent for the duplexes (5–15
times lower quantum yield) compared to the corresponding
single-strands. It should be noted that the excitation light
intensity used to promote the photodegradation of the sam-
ples in this work was deliberately excessive, to accommodate
the quantification of the photodegradation quantum yields
on a reasonable time-scale (a measurement not longer than
a few hours). In fact, as a comment on practical usability, a
trivial adjustment of excitation and emission slits, as well as
integration time in a fluorimeter setup allows for the con-
secutive acquisition of hundreds of spectra without notable
photodegradation.
The average fluorescence lifetime, <τ> of 2CNqA, like
the fluorescence quantum yield, is comparably insensitive to
the sequence surroundings in double strands. As for most
fluorescent base analogues inside nucleic acids, more than
one lifetime is needed to fit the decay data properly. For
2CNqA two or three lifetimes are required to get an accu-
rate fit to the experimental data. The average lifetimes are
fairly long compared with many fluorophores of this class
(21,22) inside DNA duplexes and vary between 7.4 ns and
10.4 ns (Table 5 and Supplementary Figure S8), which is
comparable to the lifetime of the 2CNqA monomer in aque-
ous solution (8.1 ns (32)). In contrast, the fluorescence life-
time of 2CNqA in single-stranded DNA depends more on
the sequence and is in the range of 2.8 ns (TT) to 10.9 ns
(AA) (Table 5 and Supplementary Figure S8). The fact that
the lifetimes vary more in single strands than in duplexes
reflects well the corresponding effect found for the fluores-
cence quantum yields (vide supra). Moreover, a consider-
able consistency between the variations in lifetime and fluo-
rescence quantum yield of single-stranded DNA sequences
can be observed (Table 5): those with a thymine neighbour,
having the lowest ꢀF values, also have the shortest lifetimes
(2.8–4.7 ns), whereas those where 2CNqA is embedded be-
tween two purines have the longest lifetimes (8.6–10.9 ns),
accompanied by the highest ꢀF values. The average lifetime
of all 16 single strands is 6.6 ns, which is slightly lower than
the corresponding value for duplexes (9.0 ns). The longer
lifetimes of 2CNqA in ss- and dsDNA compared to pre-
viously reported fluorescence base analogue probes with
high emission inside nucleic acids (31,43) could be useful
for biomolecule size determination experiments using fluo-
rescence anisotropy of slightly larger size ranges than what
is presently possible.
Finally, the calculated radiative decay rate constant, kR
of 2CNqA is between 3.6 and 4.3 × 107 s−1 for ssDNA se-
quences and between 3.0 and 3.4 × 107 s−1 for dsDNA se-
quences (Table 5). These low variations of kR in different en-
vironments are also reflected in a fairly stable low-energy ab-
sorption band molar absorptivities of 2CNqA inside single-
and double-stranded DNA (Table 5). The non-radiative de-
cay rate constant, kNR ranges between 0.52 and 3.2 × 108
s−1 for the 2CNqA in ssDNAs and between 0.70 and 1.0
× 108 s−1 in dsDNAs. These values can be compared with
the corresponding values for the 2CNqA monomer that are
5.2 × 107 s−1 and 0.7 × 108 s−1 for kR and kNR, respec-
tively. As can be expected from trends in quantum yields, the
non-radiative rate constant varies significantly more in the
single-stranded case compared with the duplex case, where
the quantum yield is fairly stable throughout the sequences.
Our study of the fluorescence properties of 2CNqA also
comprises an evaluation of its photodegradation quan-
tum yield (ꢀPd,D) in DNA and RNA (Supplementary Fig-
ure S10). In single-stranded DNA (ꢀPd,D = 1.4 0.05%)
2CNqA is more photostable than in single-stranded RNA
FRET between 2CNqA and qAnitro in dsDNA
We have previously reported on interbase FRET pairs con-
sisting of a cytosine donor and acceptor (33) as well as
adenine donors and an acceptor (28,30). Here, we investi-
gate a novel adenine-adenine FRET pair, 2CNqA-qAnitro
,
in DNA, and also a new interbase FRET pair between ana-
logues of two different nucleobases, 2CNqA-tCnitro, inside
RNA.
For FRET to occur, the donor and acceptor need a sig-
nificant spectral overlap. It can be seen in Figure 5, left,
that there is a large spectral overlap between the 2CNqA
emission and the qAnitro absorption. The value of the spec-
tral overlap integral, JDA,Exp = 1.6 × 1014 M−1 cm−1 nm4
(Table 6) derived from the 2CNqA-dsDNA emission and
the qAnitro-dsDNA absorption spectra (Figure 5, left) sug-
gests that the two bases constitute a useful FRET pair
for studying DNA. The spectral overlap is larger for this
FRET pair than for all DNA interbase FRET pairs that
have been reported so far (28,30,33,34), suggesting that
longer distances in duplex DNA can be investigated with
this new FRET pair. To experimentally examine the FRET
efficiency, EFRET,Exp, for the 2CNqA-qAnitro pair in DNA,
three ssDNA donor sequences (33 nt) with 2CNqA in po-
sition 7, 9 and 11, and with adenines as nearest neigh-
bours (DNA-D7, DNA-D9 and DNA-D11; Table 2), and
four complementary ssDNA acceptor sequences containing
qAnitro in position 13, 14, 19 and 20 (DNA-A13, DNA-A14,
DNA-A19 and DNA-A20; Table 2) were synthesised. By
annealing all possible combinations of these single strands,
twelve duplexes were obtained, covering the separations be-
tween the donor and the acceptor from 2–13 base pairs.
The DNA-D7:DNA-A0, DNA-D9:DNA-A0 and DNA-
D11:DNA-A0 duplexes, containing the complementary un-
modified acceptor-less strand (DNA-A0; Table 2), were
used as references (no-FRET samples). The fluorescence
quantum yield, ꢀF,D , of 2CNqA at the three donor posi-
tions is 0.23 on average. This is lower than what was found
for the dsDNA AA (0.31; Table 5) and indicates that other
factors such as small local structural changes around the
donor not resulting from the immediate neighbours could
affect the quantum yield. The FRET efficiency between
2CNqA and qAnitro in dsDNA was determined as a func-
tion of donor–acceptor separation (in bp) employing two
(ꢀPd,D = 2.61
0.10%). However, when comparing the

10 Nucleic Acids Research, 2020
Figure 5. Left: The spectral overlap between 2CNqA emission (black line; λ
= 355 nm) and qA
absorption (red line) in dsDNA. Measured in 13
nitro
Exc
mM sodium phosphate buffer, 100 mM NaCl, pH 7.5 at RT. Right: The spectral overlap between 2CNqA emission (black line; λExc = 354 nm) and tCnitro
absorption (red line) in dsRNA. Measured in 10 mM sodium phosphate buffer, 100 mM NaCl, 1.0 mM EDTA, pH 7.4 at RT. For both DNA and RNA,
the spectra are normalised at their long-wavelength maxima.
× 1014 M−1 cm−1 nm4 and for a phase angle (i.e. the angle
Table 6. The spectral overlap integral of the 2CNqA-qAnitro FRET pair in
dsDNA and of the 2CNqA-tCnitro FRET pair in dsRNA from experiment
between the donor and acceptor transition dipole moments
(JDA,Exp) and theory (JDA,The
)
at 0 bp separation) equal to 46◦. Using time-dependent den-
sity functional theory (TDDFT) to calculate the orienta-
tion of the transition dipole moment of 2CNqA and apply-
ing that in pair with the previously calculated orientation
of the transition dipole moment of qAnitro (30) both being
present in a B-DNA double helix, resulted in a phase angle
of 36◦. The agreement between computationally and exper-
imentally determined values for the spectral overlap inte-
gral and the phase angle is good and similar to previous re-
JDA,Exp
[M−1 cm−1
nm4]
JDA,The
[M−1 cm−1
nm4]
Donor
Acceptor Oligonucleotide
2CNqA
qAnitro
tCnitro
dsDNA
dsRNA
1.6 × 1014
1.9 × 1014
2.8 × 1014
2.3 × 1014
independent techniques: steady-state fluorescence measure-
ment of the decrease in the 2CNqA donor emission and
time-resolved fluorescence measurement of the shortening
of the 2CNqA donor lifetime. The final experimental FRET
efficiency, EFRET,Exp, (Figure 6, left, and Supplementary Ta-
ble S3) was obtained as a weighted average of the steady-
state (EFRET,SS; Supplementary Table S3) and time-resolved
(EFRET,TR; Supplementary Table S3) FRET efficiencies. As
can be seen in Figure 6, left, the EFRET,Exp of 2CNqA-qAnitro
in dsDNA is high at short donor–acceptor distances (2–4
bp), but it sharply decreases at about 5 bp separation, and
then oscillates between higher and lower values at longer
distances. Such a behaviour is typical for rigidly stacked in-
terbase FRET-pairs, has been seen in previous investiga-
tions (28,30,33,34) and is due to that both the distance and
the orientation between the two base analogues change with
the increasing number of separating bases. The FRET ef-
ficiencies measured at longer separations are higher than
reported for previous interbase FRET pairs (28,30,33,34).
This is an expected effect due to the larger overlap integral
for our new FRET pair and the high fluorescence quantum
yield of the donor.
ports (28,30,33). Overall, the fit between theory (EFRET,The
)
and experimentally determined FRET efficiencies is excel-
lent, except for the very short distances (Figure 6, left; base
separations 2–4). A similar trend has been observed before
(28,30,34) and is most likely an effect of FRET theory be-
ing increasingly insufficient to describe the complete en-
ergy transfer processes at these short distances; other short-
range effects like Dexter energy transfer will have an in-
creased impact as the separation between the donor and
acceptor shortens. Overall, we find that our new interbase
FRET pair, 2CNqA-qAnitro, is a useful tool for DNA stud-
ies. With the stable and high quantum yield of 2CNqA in
various DNA sequence surroundings and the large spectral
overlap with its FRET acceptor, enabling long-distance in-
terbase FRET measurements, 2CNqA-qAnitro is the most
powerful interbase FRET pair reported for DNA so far.
FRET between 2CNqA and tCnitro in dsRNA
Next, the photophysical properties of 2CNqA for the de-
velopment of a new interbase FRET system in RNA were
investigated. There is a significant spectral overlap between
the 2CNqA donor and the non-fluorescent acceptor used
in this RNA study, tCnitro (Figure 5, right). The JDA,Exp be-
An in-house designed MATLAB script was developed
and used to fit a theoretical curve, EFRET,The (bp) (see SI for
details; Supplementary Figure S11), to the experimentally
determined EFRET,Exp values (Figure 6, left). The optimal fit
was obtained for the spectral overlap integral, JDA,The of 2.8
tween them in dsRNA equals to 1.9 × 1014 M−1 cm−1 nm4

Nucleic Acids Research, 2020 11
Figure 6. Left: Experimental FRET efficiency (EFRET,Exp; dots) as a function of the number of base-pairs separating the 2CNqA donor and qAnitro acceptor
in dsDNA compared with the theoretical FRET efficiency (EFRET,The; line). Measured in 13 mM sodium phosphate buffer, 100 mM NaCl, pH 7.5 at RT.
Right: Experimental FRET efficiency (EFRET,Exp; dots) as a function of the number of base-pairs separating the 2CNqA donor and tCnitro acceptor in
dsRNA compared with the theoretical FRET efficiency (EFRET,The; line). Measured in 10 mM sodium phosphate buffer, 100 mM NaCl, 1.0 mM EDTA,
pH 7.4 at RT. For both DNA and RNA, the experimental values are weighted averages of FRET efficiencies determined in steady-state (EFRET,SS) and
time-resolved (EFRET,TR) fluorescence experiments. The error bars represent the weighted variance of EFRET,Exp. The EFRET,The depicted as a line shows
the predicted values also at unnatural non-integer separations.
(Table 6), which is slightly higher than for the DNA system
presented above.
A-form RNA model and the photophysical properties of
the probes as an input. The best overlap between the cal-
culations and the experimental data was obtained for the
spectral overlap integral, JDA,The, of 2.3 × 1014 M−1 cm−1
nm4 and an angle between the 2CNqA transition dipole
and the X-axis (tD) set to 45.5◦. From TDDFT calcula-
tions this angle is predicted to be 27.5◦. Hence, the agree-
ment between JDA from spectra and from the FRET data
is good. Additionally, the TDDFT-predicted orientation of
the 2CNqA transition dipole moment fits satisfactorily with
what is suggested by the FRET measurements. Compared
with in DNA, the theoretical FRET values fit better to
experimental values at shorter distances in RNA (Figure
6, right). Overall, our new interbase FRET pair, 2CNqA-
tCnitro, performs excellently in the RNA system studied and
preserves the A-form secondary structure. Given the quan-
tum yield of 2CNqA in RNA and the large spectral over-
lap with its FRET acceptor, the 2CNqA-tCnitro interbase
FRET pair constitutes a valuable complement to the tCO-
tCnitro FRET pair (45) – the only FRET pair studied in
RNA to date, which is limited to cytosine substitutions, has
a lower spectral overlap but a higher donor quantum yield
than the FRET pair we report herein.
To study the FRET efficiency at different separations
of the 2CNqA and tCnitro inside RNA, four 2CNqA-
containing donor sequences (RNA-D6, RNA-D7, RNA-
D12 and RNA-D13 with adenines as nearest neighbours
for all donor strands; Table 3) with three complementary
tCnitro-containing acceptor sequences (RNA-A6, RNA-A8
and RNA-A10; Table 3) were synthesised and hybridized,
resulting in twelve duplexes with 2–13 base pairs separating
the donor and acceptor. As no-FRET reference duplexes,
RNA-D6:RNA-A0, RNA-D7:RNA-A0, RNA-D12:RNA-
A0 and RNA-D13:RNA-A0, each containing the comple-
mentary acceptor-less strand (RNA-A0; Table 3) were used.
The average fluorescence quantum yield of the no-FRET
RNA duplexes is 0.12 which is lower than the correspond-
ing value of the DNA FRET study above (0.23) and indi-
cates a lower overall quantum yield of 2CNqA in RNA than
in DNA systems, possibly as an effect of their different sec-
ondary structures. The donor–acceptor FRET efficiencies
in dsRNA as a result of various base separations are pre-
sented in Figure 6, right, and Supplementary Table S4. The
EFRET,Exp is close to the maximum value (1.0) for duplexes
in which the acceptor is in the close proximity to the donor
(2–4 bp), but at larger separations it significantly changes
due to an increase in distance and also varies in a periodical
fashion due to the change in orientation factor, similar to
the DNA system. These data suggest that both the 2CNqA
and tCnitro bases are firmly stacked inside the RNA. More-
over, and as in the case reported for DNA above, this sug-
gests the great applicability of 2CNqA in RNA interbase
FRET experiments.
CONCLUSIONS
We have here performed a thorough photophysical and
biophysical study of a new fluorescent adenine analogue,
2CNqA, inside DNA and RNA. Using circular dichroism,
duplex melting and mismatch investigations, we show that
2CNqA is an excellent analogue of adenine in both DNA
and RNA contexts. In the photophysical characterisation,
we demonstrate that 2CNqA retains a high quantum yield
inside nucleic acids. Unlike our previously reported bright
adenine analogues, qAN1 and pA, the quantum yield of
Using FRETmatrix (44), the expected FRET efficiency
between 2CNqA and tCnitro at different separations can be
calculated (line in Figure 6, right). The software uses a static

12 Nucleic Acids Research, 2020
2CNqA in duplex DNA is high irrespective of sequence
context, and we find 2CNqA to be the brightest single-
photon excitation fluorescent base analogue inside DNA
reported until now. These powerful spectroscopic proper-
ties can be combined with our previously reported inter-
base FRET acceptors, qAnitro and tCnitro, to constitute ex-
cellent FRET pairs in DNA and RNA, respectively. Thanks
to a large spectral overlap and high donor quantum yield,
2CNqA and qAnitro constitute the most powerful FRET
pair reported for DNA so far. We envision that our new
2CNqA adenine analogue, due to its significant brightness,
is a promising alternative to external fluorophores as an in-
ternal label for nucleic acids. Finally, we anticipate its use in
a significant number of applications as an interbase FRET
donor in pair with our acceptors reported herein; for ex-
ample, we are currently utilizing it in assays reporting on
interactions between drug molecules and pre-miRNAs.
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FUNDING
European Union’s Horizon 2020 research and innovation
programme under the Marie Skłodowska-Curie Actions In-
dividual Fellowship [H2020-MSCA-IF-2016, grant agree-
ment no. 753595 to A.W.d.N.]; Swedish Foundation for
Strategic Research [SSF, grant no. IS14-0041 and IRC15-
0065 to L.M.W., ID14-0036 to M.G.]; Swedish Research
Council [VR, grant no. 2017-03707 to L.M.W.]. Funding for
open access charge: VR (The Swedish Research Council).
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