A Highly Fluorescent Nucleobase Molecular Rotor

Article abstract of DOI:10.1021/jacs.0c05180

Fluorescent base analogs (FBAs) are powerful probes of nucleic acids' structures and dynamics. However, previously reported FBAs exhibit relatively low brightness and therefore limited sensitivity of detection. Here we report the hitherto brightest FBA that has ideal molecular rotor properties for detecting local dynamic motions associated with base pair mismatches. The new trans-stilbene annulated uracil derivative tsT exhibits bright fluorescence emissions in various solvents (? × φ = 3400-29700 cm-1 M-1) and is highly sensitive to mechanical motions in duplex DNA (? × φ = 150-4250 cm-1 M-1). tsT is thereby a smart thymidine analog, exhibiting a 28-fold brighter fluorescence intensity when base paired with A as compared to T or C. Time-correlated single photon counting revealed that the fluorescence lifetime of tsT (τ = 4-11 ns) was shorter than its anisotropy decay in well-matched duplex DNA (θ = 20 ns), yet longer than the dynamic motions of base pair mismatches (0.1-10 ns). These properties enable unprecedented sensitivity in detecting local dynamics of nucleic acids.

Full text of DOI:10.1021/jacs.0c05180

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A Highly Fluorescent Nucleobase Molecular Rotor
Ashkan Karimi, Richard Börner, Guillaume Mata, and Nathan W. Luedtke
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c05180 • Publication Date (Web): 11 Aug 2020
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A Highly Fluorescent Nucleobase Molecular Rotor
Ashkan Karimia,b, Richard Börnera,c, Guillaume Mataa, Nathan W. Luedtkea,b,d
*
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a Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland
b Department of Chemistry, McGill University, H3A-0B8 Montreal, Canada
c Laserinstitut Hochschule Mittweida, University of Applied Sciences, 09648 Mittweida, Germany
d Department of Pharmacology and Therapeutics, McGill University, H3A-1A3 Montreal, Canada
Supporting Information Placeholder
attempts have thus far resulted in either bright FBAs with low
ABSTRACT: Fluorescent base analogs (FBAs) are powerful
probes of nucleic acids’ structures and dynamics. However,
previously reported FBAs exhibit relatively low brightness and
therefore limited sensitivity of detection. Here we report the
hitherto brightest FBA that has ideal molecular rotor
properties for detecting local dynamic motions associated with
base pair mismatches. The new trans-stilbene annulated uracil
derivative “tsT” exhibits bright fluorescence emissions in
sensitivity towards SNPs,15-19 or highly sensitive FBAs with low
brightness.8,20,21 Here we present the brightest and most
sensitive FBA-mismatch reporter to date. The trans-stilbene-
containing thymidine analog “tsT” acts as an ideal molecular
rotor for SNP detection,22-27 with a fluorescent lifetime (τ = 4 –
11 ns) that is slightly longer as compared to timescale of local
dynamic motions of base pair mismatches (0.1 – 10 ns).28-30
This provides unprecedented sensitivity and specificity for
detecting a single adenine residue in the opposing strand, with
a ~20-fold brighter fluorescence intensity for matched “A” (Ԑ
various solvents (Ԑ
Φ = 3,400 – 29,700 cm-1 M-1) and is
×
highly sensitive to mechanical motions in duplex DNA (Ԑ
Φ
×
= 150 – 4,250 cm-1 M-1). tsT is thereby a “smart” thymidine
analog, exhibiting a 28-fold brighter fluorescence intensity
when base paired with A as compared to T or C. Time-
correlated single photon counting revealed that the
fluorescence lifetime of tsT (τ = 4 – 11 ns) was shorter than its
anisotropy decay in well-matched duplex DNA (θ = 20 ns), yet
longer than the dynamic motions of base pair mismatches (0.1
– 10 ns). These properties enable unprecedented sensitivity in
detecting local dynamics of nucleic acids.
Φ = 3,000 – 4,250 cm-1 M-1) versus mismatched “C, T, and
×
×
G” bases (Ԑ
Φ = 150 – 520 cm-1 M-1).
Fluorescent nucleobase analogs (FBAs) provide powerful
tools for probing nucleic acids’ structures, dynamics and
binding interactions.1,2 Compared to conjugated fluorophores
and intercalating dyes, FBAs have the advantage of highly
precise positioning within RNA and DNA structures. However,
fluorescence quenching of FBAs by neighboring residues via
photo-induced electron transfer (PET) can dramatically limit
their brightness.3-6 For example, small electron-poor systems
such as 2-aminopurine (2AP) are reductively quenched by
purines,7 and electron-rich systems such as MDA are oxidatively
quenched by pyrimidines.8 The brightest, previously reported
Figure 1. Average brightness of selected FBAs in well-
matched duplex DNA (cm-1 M-1) and their calculated HOMO-
LUMO energy levels (kcal mol-1). DFT calculations (where R =
Me) were performed using B3LYP/6-31+G(d) in water (see
Table S1 and Figure S1 for details). The dashed lines represent
the LUMO level of the most easily reduced nucleobase (T), and
the HOMO level of the most easily oxidized base (G).31
fluorescent nucleobase analogs, such as tCo (Ԑ
Φ ≈ 2,000 cm-
×
1 M-1),9,10 avoid PET quenching by having HOMO-LUMO energy
levels that are intermediate between the HOMO of guanine and
the LUMO of thymidine (Figure 1 and Figure S1). However, the
fluorescence intensity of tCo and related analogs exhibit little-
to-no environmental sensitivity and therefore have limited
utility as reporters of base pair mismatches.11,12 Given the
growing clinical interest in point-of-care detection of single
nucleotide polymorphisms (SNPs),13 there have been
numerous efforts towards the development of SNP sensors
based on FBA-mismatch detection.8,14-27 However, these
Design of the new fluorescent thymidine analog “tsT” was
inspired by the excellent base pairing specificities of 6-
19,32-34
substituted quinazolines,
together with the desirable
photophysical properties of trans-stilbene that include
viscosity-sensitive emissions.35 Density Functional Theory
(DFT) calculations predicted that a ring-fused, uracil-trans-
stilbene derivative “tsT” should not undergo fluorescence
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quenching via PET (Figure 1). We therefore commenced
tsT increased with increasing viscosity (Figure 2A and Figure
S3), consistent with rotation-induced fluorescent quenching.
Encouraged by these results, we synthesized tsT
phosphoramidite 3 for its incorporation into DNA. Nucleoside
2 was treated with DMTCl and chlorophosphoramidite to
obtain >500 mg of pure tsT phosphoramidite 3. The entire 11-
step sequence required only 7 chromatographic separations
and provided 3 in a 26% overall isolated yield.
synthesis of tsT by preparing 6-bromo-quinazoline-2,4-(3H)-
dione nucleoside 1 as a single diastereoisomer on a multi-gram
scale in a 46% overall yield (7 steps) according to our
previously established protocol.36 Suzuki-Miyaura coupling,
followed by silyl deprotection furnished tsT nucleoside 2 in a
92% isolated yield over 2 steps (Scheme 1 and Scheme S1).
1
2
3
4
5
6
7
8
Using standard, solid-phase supported synthesis,39 tsT was
incorporated into three different oligonucleotide DNAs (ODN1
Scheme 1. Synthesis of tsT nucleoside (2) and tsT
phosphoramidite (3)a ,b
.
9
–
ODN3). Following their purification and annealing to
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complementary strands, canonical B-form secondary
structures were observed using circular dichroism (Figure S6).
The maximum absorbance of tsT remains very strong in DNA
±
O
N
O
Br
Br
NH
NH
7 steps
a)
b)
O
N
O
(Ԑ310nm =30,600
700 cm-1 M-1) and sufficiently red shifted to
H
+
O
AcO
TIPSO
have no overlap with the natural bases (Fig S7A). The
O
OAc
brightness of the three duplexes containing a tsT:A base pair (Ԑ
OTBS
Φ = 3,000 – 4,250 cm-1 M-1) were about the same as the free
×
AcO
(1)
nucleoside 2 in PBS buffer (3,420 cm-1 M-1), consistent with a
lack of tsT fluorescence quenching by neighboring residues via
PET. Thermal denaturation experiments revealed that tsT
exhibits similar base pairing specificity as thymidine,40 with
the temperature of ODN1 duplex melting (Tm) being tsT:C/T <
tsT:G < tsT:A (Table 1 and Figure S8). The brightness of tsT was
O
N
O
N
NH
NH
c)
d)
O
O
O
O
CN
HO
DMTO
O
×
much more sensitive to this same trend, with Ԑ
Φ values
O
P
OH
tsT (2)
N
ranging from 150 – 4,250 cm-1 M-1 for tsT:C/T < tsT:G << tsT:A
(Table 1, Figure 2B and Figure S7B). Similar trends were also
observed for the relative broadness of thymidine imino proton
resonances in 1H NMR spectra of T:G and T:T base pairs in
duplex DNA.41 Together these results suggested that
fluorescence quenching of tsT is related to increased molecular
motions of mismatched tsT:C/T base pairs over both tsT:G
(wobble) and tsT:A base pairs.
(3)
> 0.5 g scale
a
Reagents and conditions: (a) trans-2-phenylvinyl boronic acid
pinacol ester (2.1 equiv.), Cs2CO3 (3.2 equiv.), Pd(dppf)Cl2, 1,4-dioxane,
Ar, 100 oC, 24 h, (98%). (b) TBAF (4.9 equiv.), THF, 25 oC, 22 h, (94%).
(c) DMTCl (1.4 equiv.), pyridine, Ar, 25 oC, 165 min, (68%). (d) 2-cyano
ethyl N, N-diisopropyl chlorophosphoroamidite (4.0 equiv.), DIPEA
(5.0 equiv.), CH2Cl2, Ar,
triisopropylsilyl, TBS
Bis(diphenylphosphino)ferrocene,
butylammoniumfluoride, THF
dimethoxytriphenylmethyl
0
to 25 oC, 150 min, (90%). TIPS
tert-butyldimethylsilyl, dppf 1,1′-
TBAF tetra-n-
tetrahydrofuran, DMTCl
chloride, DIPEA
=
=
=
=
Table 1: Fluorescence quantum yield (Φ), brightness
=
=
4,4′-
N,N-
×
(Ԑ
Φ) and melting temperature (Tm) of double-
=
stranded or single-stranded (ss) DNA.a
b
diisopropylethylamine.
See the SI for synthetic details and
×
Ԑ
Φ
As compared to trans-stilbene itself (Φ = 0.02 – 0.04), the ts
nucleoside 2 exhibits a similar extinction coefficient (Ԑ310nm
T
=
Oligonucleotide
Φ
Tm (oC)
(cm-1 M-1)
4,250
520
±
36,500
5,600 cm-1 M-1), yet much larger quantum yield (Φ =
tsT:A
tsT:G
55.0 c
53.0
51.0
50.8
-
0.139
0.017
0.005
0.005
0.028
0.098
0.012
0.020
0.134
0.026
0.11 – 0.75) in various solvents (Table S2). Decreasing
quantum yields and increasing Stokes’ shifts of nucleoside 2
were observed with increasing solvent polarity (Figure S2).
These results are consistent with formation of a twisted
intramolecular charge transfer (TICT) excited state leading to
ODN1b
tsT:C
tsT:T
ss-tsT
tsT:A
tsT:T
ss-tsT
tsT:A
ss-tsT
150
non-radiative decay.37,38 To further evaluate the potential of ts
T
150
as
a fluorescent molecular rotor, the absorption and
fluorescence emission of nucleoside 2 was measured in various
mixtures of methanol and glycerol. These solvents have very
similar polarities yet drastically different viscosities. The
quantum yield of
860
3,000
370
60.2 d
56.1
-
ODN2b
ODN3b
610
4,100
800
54.1e
-
a All measurements were performed at 25 oC in PBS buffer (pH = 7.4),
b
with DNA concentrations
=
2.0 μM.
ODN1 sequence: 5’-
GCGTAtsTCGTATACAC-3’. ODN2 sequence: 5’-GCGTAtsTGCGTATACAC-
3’. ODN3 sequence: 5’-GCGTAtsTATGTATACAC-3’.
Tm of the
c
Figure 2. (A) Fluorescence of tsT nucleoside 2 in mixtures of
methanol and glycerol; and (B) Fluorescence of tsT
functionalized duplex DNA (ODN1) containing matched or
mismatched base pairs in PBS buffer (pH = 7.4). All emission
spectra were collected using an excitation wavelength of 310
corresponding unmodified duplex = 49.7 oC. Tm of the unmodified
d
duplex DNA = 56.3 oC. e Tm of the unmodified duplex DNA = 49.3 oC.
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±
7.9
1.7
ss-tsT
25
±
±
1.98 0.14
5.4
0.1
1
2
3
4
5
6
7
8
To gain insight into the relationships between tsT dynamic
motions and brightness in DNA, time-correlated single photon
counting (TCSPC) experiments were conducted using ds-tsT:A,
a All measurements were performed using 2.0 μM of DNA in PBS
ds-tsT:T and ss-tsT samples of ODN1 (Table 2). All time-resolved
buffer (pH = 7.4). All values reflect averages and standard deviations
data fit to single-exponential decay models (R2 = 0.98 – 0.99,
of three measurements. b See Table S5 for details. c Calculated from
o
d
Eq. S2 and Figure S9). At 25 C, the fluorescence lifetime values
θ (Eq. S5). Value reflects local dynamics, not RH.
To evaluate local and global dynamic motions, TCSPC
experiments were used to measure time-resolved fluorescence
anisotropy decay (θ, Figure 3B, Eq. S4). At 25 oC, the θ values of
tsT in DNA followed the same trends as steady-state quantum
yields and fluorescence lifetime measurements, where ds-tsT:A
(θ = 20 ns) >> ss-tsT (θ = 7.9 ns) > ds-tsT:T (θ = 5.3 ns). The θ
(τ) of tsT in DNA were ds-tsT:A (11 ns), ss-tsT (5.4 ns), and ds-
tsT:T (4.5 ns), reflecting the same trends as their steady-state
brightness. Upon heating each sample over a “pre-melting”
temperature range of 25 oC  45 oC where no global melting of
the duplexes was observed, the τ values of tsT decreased in a
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linear fashion for both ds-tsT:A (-0.11 ns per C) and ss-tsT (-
o
o
o
values of ds-tsT:A at 25 C and 35 C were essentially identical
(θ = 19 – 20 ns, Table 2 and Figure S10), suggesting that the θ
values are reflecting global motions of the duplex at these
temperatures. We therefore used the θ value at 25 oC to
calculate the hydrodynamic radius of the duplex (RH, Eq. S5),
giving RH = 2.70 ± 0.18 nm (Table 2). This value is in full
agreement with duplex DNA RH values previously measured
using fluorescence anisotropy, sedimentation velocity and
Monte Carlo simulations.9,45,46 At 55 oC the θ of ds-tsT:A (8.5 ns)
approached that of ss-tsT at 25 oC (7.9 ns). These values are
consistent with the conversion of a “rod-like” duplex (RH = 2.70
nm) into a “worm-like” chain of ssDNA (RH = 2.09 nm, Table
2).47,48 The close agreements between our results obtained
using tsT with the global physical properties of ssDNA,49,50
suggest that ssDNA contains partially-ordered nucleobases
with correlation times dominated by concerted, global motions
of the entire molecule. In contrast, the θ for mismatched,
duplex ds-tsT:T (5.3 ns) primarily reflects rapid, local dynamics,
since this value is much lower than that of ds-tsT:A (20 ns), and
0.05 ns per oC, Figure 3A). These results are consistent with
increased “breathing” motions of nucleobases with increasing
temperatures.42 Surprisingly, the τ values of ds-tsT:T increased
over this same temperature range (+0.03 ns per oC), suggesting
increasing rigidity of the mismatch with increasing
temperature. This result is consistent with the higher affinity
and positive entropy change exhibited by T:T mismatches for
HgII binding over these temperatures.43,44 At 55 oC the τ values
for all three samples neared convergence due to thermal
melting of the duplexes (Figure 3A).
o
it is even lower than ss-tsT at 25 C (7.9 ns). These results are
consistent with previous NMR measurements and molecular
dynamics simulations suggesting the presence of rapid, local
motions of base pair mismatches in otherwise canonical
duplexes.28 To our knowledge this is the first time that an FBA
has been used to determine the hydrodynamic radii of single-
stranded DNA, and our results suggest that the dynamic
motions of a base pair mismatch in duplex DNA are even
greater than those present in single-stranded DNA.
Figure 3. (A) Temperature-dependent fluorescence lifetime
values (averaged values of three measurements with standard
errors connected by solid lines) compared to global thermal
melting according to absorbance changes (O.D., dashed lines)
at 260 nm. (B) Time-resolved fluorescence anisotropy of ss-tsT,
ds-tsT:T and ds-tsT:A (ODN1) at 25 °C. All measurements were
conducted in PBS buffer (pH = 7.4).
In summary, tsT is the brightest and most environment
×
sensitive FBA reported to date (Ԑ
Φ = 150 – 29,700 cm-1 M-
1). The fluorescence lifetime of tsT (τ = 4 – 11 ns) is longer than
the time scale of local dynamic motions of base pair
mismatches (0.1 – 10 ns),28-30 yet faster than the global
anisotropy decay of tsT in duplex DNA (θ = 20 ns). These
properties together enable unprecedented sensitivity and
specificity in the detection of an adenine residue in the
complement strand. In addition to highly sensitive base pair
match/mismatch discrimination, tsT has provided insights into
the fundamental dynamic behavior of duplex and single-
stranded DNA using TCSPC experiments. The exceptional
brightness and sensitivity of tsT may enable other types of
demanding applications, such as tracking of single-molecule
dynamics.51
Table 2. Fluorescence lifetime (τ), anisotropy decay
(θ), and hydrodynamic radius (RH) of ODN1 double-
stranded or single-stranded (ss).a, b
T (oC)
τ (ns)
θ (ns)
RH (nm)c
±
0.1
±
20
4.2
11.1
±
25
2.70
0.18
tsT:A
tsT:T
±
0.1
10.0
35
55
25
19 ± 3.8
8.5 ± 2.8
2.68 ± 0.18
2.09 ± 0.22
±
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Description of materials and
methods, computational details, synthetic procedures,
compounds characterization and NMR spectra, circular
±
±
6.6
4.5
0.1
0.1
±
5.3
1.6
1.74
0.17d
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dichroism (CD), thermal denaturation (Tm) and fluorescence
spectra, Scheme S1, Figures S1-S10 and Tables S1-S5.
1
2
3
4
5
6
7
8
9
13. Xu, H.; Xia, A.; Wang, D.; Zhang, Y.; Deng, S.; Lu, W.; Luo, J.; Zhong, Q.;
Zhang, F.; Zhou, L.; Zhang, W.; Wang, Y.; Yang, C.; Chang, K.; Fu, W.; Cui,
J.; Gan, M.; Luo, D.; Chen, M., An ultraportable and versatile point-of-
care DNA testing platform. Science Advances 2020, 6 (17), eaaz7445.
AUTHOR INFORMATION
Corresponding Author
Email: nathan.luedtke@mcgill.ca
14. Hwang, G. T., Single-Labeled Oligonucleotides Showing
Fluorescence Changes Upon Hybridization with Target Nucleic Acids.
Molecules 2018, 23 (1), 124.
Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENT
We thank Prof. R. K. O. Sigel for his support of R.B. We thank the
University of Zurich, McGill University, University of Applied
Sciences Mittweida, the Swiss National Science Foundation
(grant No. 165949 to N.W.L), the National Research Council of
Canada (grant No. 05048 to N.W.L), and the McGill Centre for
Structural Biology (studentship to A.K.) for funding.
16. Burns, D. D.; Teppang, K. L.; Lee, R. W.; Lokensgard, M. E.; Purse, B.
W., Fluorescence Turn-On Sensing of DNA Duplex Formation by a
Tricyclic Cytidine Analogue. J Am Chem Soc 2017, 139 (4), 1372—1375.
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M. E.; Cooksy, A. L.; Purse, B. W., Electronic Modifications of Fluorescent
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