Synthesis and biophysical studies of coronene functionalized 2′-amino-LNA: A novel class of fluorescent nucleic acids

Article abstract of DOI:10.1021/bc900421r

Incorporation of 2′-N-(coronen-1-yl)methyl-2′-amino-LNA monomer X or 2′-N-4-(coronen-1-yl)-4-oxobutanoyl-2′-amino-LNA monomer Y into short DNA strands induces high binding affinity toward DNA or RNA and a marked, red-shift in steady-state fluorescence emi

Full text of DOI:10.1021/bc900421r

Bioconjugate Chem. 2010, 21, 513–520
513
Synthesis and Biophysical Studies of Coronene Functionalized
2′-Amino-LNA: A Novel Class of Fluorescent Nucleic Acids
Pankaj Gupta, Niels Langkjær, and Jesper Wengel*
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark.
Received September 23, 2009; Revised Manuscript Received December 27, 2009
Incorporation of 2′-N-(coronen-1-yl)methyl-2′-amino-LNA monomer X or 2′-N-4-(coronen-1-yl)-4-oxobutanoyl-
2′-amino-LNA monomer Y into short DNA strands induces high binding affinity toward DNA or RNA and a
marked red-shift in steady-state fluorescence emission upon hybridization to cDNA or RNA.
INTRODUCTION
not a common fluorescent dye for modification of oligonucle-
otides, whereas it has been used in the field of lipids (55). Nagata
et al. (56) described coronene as an external binder of DNA,
but later, Zinger et al. (57) described that the planner coronene
molecules partially insert between adjacent DNA bases. Herein,
we report the synthesis of monomer X (with a methylcoronene
moiety attached to 2′-amino-LNA) and monomer Y (with a
oxobutanoylcoronene moiety attached to 2′-amino-LNA) and
oligonucleotides involving them, together with their hybridiza-
tion and fluorescence properties.
Fluorescent labeling of nucleosides and oligonucleotides is
explored for structural studies, nucleic acid sequencing, mo-
lecular diagnostics, and other applications relating to genomics
(1). “Molecular beacons” (2-4), fluorophore-quencher com-
bination probes (5), “scorpion” (6), “Pacman” (7) or “sunrise
primers,” (8) specific pyrene-conjugated DNA or RNA probes
(9-24), and fluorescence resonance energy transfer (FRET)
probes (5, 25, 26) are typical example of such probes. Other
fluorescent probes have been designed by labeling of oligonucle-
otides with a single hybridization sensitive moiety (17, 27-37).
These methods have several potential advantages such as
sensitivity, flexibility, safety, and relative inexpensiveness
compared to the classical radiolabeling method.
The high-affinity hybridization of LNA (locked nucleic
acid) (37-42), 2′-amino-LNA (43-45), and R-L-LNA (46) and
the properties of LNA modified oligonucleotides as diagnostic
tools and therapeutic agents (47, 48) encouraged us to appraise
N-functionalized derivatives of 2′-amino-LNA. Recently, LNAs
have been functionalized by fluorescent groups such as
pyrene (15, 18, 49, 50), perylene (51, 52), and (phenylethy-
nyl)pyrene (53). These pyrene-functionalized LNAs exhibit
appealing properties and can be used to sense full complemen-
tarity of DNA or RNA counterparts.
Recently, Hrdlicka et al. (18) investigated multilabeled
pyrene-functionalized 2′-amino-LNA probes which exhibited
high-affinity hybridization to DNA/RNA complements, quenched
fluorescence of single-stranded probe, and large increase of the
quantum yield and fluorescence brightness upon hybridization.
More recently, Astakhova et al. (52, 53) reported multilabeled
perylene- and (phenylethynyl)pyrenecarbonyl-functionalized 2′-
amino-LNA probes which exhibited high-affinity hybridization
toward cDNA/RNA, considerably quenched fluorescence of
single-stranded multilabeled probes, increased fluorescence
brightness upon hybridization, high fluorescence quantum yields,
and red-shifted emission compared to pyrene-functionalized 2′-
amino-LNA probes. However, there is still a need to find optimal
fluorochromes which, e.g., display a long-wave emission
spectrum relative to pyrene, perylene, and (phenylethynyl)pyre-
necarbonyl.
EXPERIMENTAL PROCEDURES
General. Anhydrous reactions were carried out under an
atmosphere of nitrogen. Solvents used were of HPLC grade.
Acetonitrile and dichloromethane were made anhydrous by
storage over molecular sieves (4 Å). TLC was run with Merck
silica 60 F254 aluminum sheets. 1H NMR spectra were recorded
at 300 MHz, 13C NMR spectra at 75.5 MHz, and 31P NMR
spectra at 121.5 MHz (δH: CDCl3 7.26 ppm, DMSO-d6 2.50;
δC: CDCl3 77.0 ppm, DMSO-d6 39.4). Chemical shifts are
reported in ppm relative to either tetramethylsilane or the
deuterated solvent as an internal standard for 1H NMR and 13
C
NMR, and relative to 85% H3PO4 as external standard for 31P
NMR. Assignments of NMR spectra, when given, are based
on 2D NMR experiments. Coupling constants (J values) are
given in Hertz. ESI-HRMS were recorded in positive ion mode
on an API QSTAR mass spectrometer.
Synthesis of ON2-ON10. Nucleoside phosphoramidites 3a
and 3b were used as building blocks for incorporation of 2′-
N-(coronen-1-yl)methyl-2′-amino-LNA monomer X and 2′-N-
(coronen-1-yl)-4-oxo-butanoyl-2′-amino-LNA monomer Y, re-
spectively, into DNA oligonucleotides (Scheme 1, Table 1) in
0.2 µmol scale synthesis using an automated DNA synthesizer.
Standard procedures were used for unmodified monomers (i.e,
3% trichloroacetic acid in CH2Cl2 as detritylation reagent; 0.25
M 4,5-dicyanoimidazole (DCI) in CH3CN as activator; acetic
anhydride in THF as cap A solution; N-methylimidazole in THF
as cap B solution; and 0.02 M iodine in H2O/pyridine/THF as
the oxidizing solution). The stepwise coupling yields were >99%
for unmodified phosphoramidites (2 min coupling time) and for
3a and 3b 95% and 99% (10 min coupling time, 1H-tetrazole
as activator), respectively, based on the absorbance of the
dimethoxytriyl cation released after each coupling step. After
deprotection and cleavage from solid support (32% aq ammonia,
55 °C, 12 h), the modified ONs were precipitated and repeatedly
washed with ethanol, and their composition and purity (>80%)
verified by MALDI-MS analysis and ion-exchange HPLC,
respectively.
Coronene is a fluorochrome which resembles pyrene in its
chemical behavior but differentiates from pyrene with respect
to photochemical properties (54). Coronene is very stable,
showing the properties of a fully benzenoid hydrocarbon. The
extra stability of coronene imparted by the sextet ring current
has been called “superaromaticity”. Unlike pyrene, coronene is
* E-mail jwe@ifk.sdu.dk.
10.1021/bc900421r  2010 American Chemical Society
Published on Web 01/25/2010

514 Bioconjugate Chem., Vol. 21, No. 3, 2010
Gupta et al.
Scheme 1a
spectrophotometer using quartz optical cells with a path length
of 1.0 cm. Measurements were conducted using 1.0 µM of
strands in 10 mM phosphate buffer ([Na+] ) 110 mM, [Cl-]
) 100 mM, [EDTA] ) 0.1 mM, pH 7.0).
(1R,3R,4R,7S)-5-[4-Coronen-1-ylmethyl]-1-(4,4′-dimethoxy-
trityloxymethyl)-7-hydroxy-3-(thymin-1-yl)-2-oxa-5-azabi-
cyclo[2.2.1]heptane (2a). Nucleoside 1 (58) (0.23 g, 0.40 mmol)
was dissolved in anhydrous 1,2-dichloroethane (5 mL). To this
stirred solution at rt were added coronene-1-carbaldehyde (0.14
g, 0.42 mmol) and sodium triacetoxyborohydride (0.12 g, 0.60
mmol). After stirring at rt for 6 h, saturated aq NaHCO3 (2 mL)
was added and the mixture diluted with ethylacetate (20 mL).
The organic phase was washed with saturated aq NaHCO3 (10
mL) and the aq phase was extracted with ethylacetate (2 × 20
mL). The combined organic phase was dried over Na2SO4,
filtered, and evaporated to dryness under reduced pressure, and
the resulting residue was purified by silica gel column chro-
matography (20-70% EtOAc in petroleum ether, v/v) to afford
nucleoside 2a as a yellowish solid material (210 mg; 60%). Rf
) 0.35 (7% MeOH in CH2Cl2, v/v); ESI-HRMS m/z 906.3127
([M+Na]+, C57H45N3O7 ·Na+ calc. 906.3149); 1H NMR (DMSO-
d6) δ (selected signals) 11.39 (s, NH), 9.16-8.91 (m, Ar), 7.70
(s, H6), 7.48-6.88 (m, Ar), 5.94 (s, 3′-OH), 5.74 (d, J ) 6.0
Hz, H1′), 5.21 (d, J ) 14.4 Hz, CH2Ar), 5.03 (d, J ) 14.7 Hz,
CH2Ar), 4.35 (d, J ) 4.5 Hz, H2′), 3.78-3.72 (m, H3′ and 2 ×
OCH3), 3.48 (d, J ) 10.8 Hz, H5′a/b), 3.38-3.35 (m, H5′a/b
overlapping with H2O), 3.18 (d, J ) 9.6 Hz, H5′′a), 2.97 (d, J
) 6.0 Hz, H5′′b), 1.55 (s, CH3); 13C NMR (DMSO-d6) δ 163.8,
158.1, 150.0, 144.7, 135.4, 135.2, 134.7, 134.0, 129.8, 129.7,
128.1, 128.0, 127.9, 127.8, 127.6, 126.9, 126.8, 126.3, 126.1,
126.0, 125.8, 122.6, 121.8, 121.6, 121.5, 121.3, 121.2, 120.8,
113.2, 108.2, 88.5, 86.2, 85.6, 70.7, 66.5, 59.7, 58.1, 56.8, 55.0,
12.4.
a Reagents and conditions: (i) (2a) Coronene-1-carbaldehyde,
NaBH(OAc)3, ClCH2CH2Cl, rt, 60%; (2b) 4-(coronen-1yl)-4-oxobu-
tanoic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDC ·HCl), CH2Cl2, 59%; (ii) bis(N,N-diisopropylamino)-2-
cyanoethoxyphosphine, N,N-diisopropylammonium tetrazolide, MeCN/
CH2Cl2 (5:2), 2 h for 3a/35 °C, 3 h for 3b/rt; 3a, 80%; 3b, 75%. T )
thymin-1-yl. DMT ) 4,4′-dimethoxytrityl.
Table 1. Thermal Denaturation Studies Towards DNA/RNAa
DNA
Tm/°C ∆Tm/°C
28.5
28.5
RNA
Tm/°C ∆Tm/°C
26.5
oligonucleotides
ON1 5′-d(GTG ATA TGC)b
ON2 5′-d(GCA TAT CAC)
ref.
ref.
ref.
24.5 ref.
28.0
24.0
27.5
18.0/37.0
ON3 5′-d(GTG AXA TGC)c,d 31.5
+3.0
-1.5
+2.0
+1.5
-0.5
+3.0
ON4 5′-d(GCA TAX CAC)
ON5 5′-d(GCA XAT CAC)
ON6 5′-d(GCA XAX CAC)
27.0
30.5
17.5/36.5
ON7 5′-d(GTG AYA TGC)c,d 43.0
+14.5 36.0
+9.0 36.5
+15.0 35.0
+1.5 19.0
+9.5
ON8 5′-d(GCA TAY CAC)
ON9 5′-d(GCA YAT CAC)
ON105′-d(GCA YAY CAC)
37.5
43.5
31.5
+12.0
+10.5
-2.7
(1R,3R,4R,7S)-5-[4-Coronen-1-ylmethyl]-7-[2-cyanoethoxy-
(diisopropylamino)-phosphinoxy]-1-(4,4′-dimethoxytrityloxy-
methyl)-3-(thymin-1-yl)-2-oxa-5-azabicyclo [2.2.1]heptane
(3a). Nucleoside 2a (0.13 g, 0.14 mmol) was dissolved in
anhydrous acetonitrile (5 mL) under stirring at rt. N,N-
Diisopropylammonium tetrazolide (0.037 g, 0.22 mmol) and
bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine (0.14 mL,
0.44 mmol) were added under nitrogen. The reaction mixture
was stirred for 3 h at 35 °C and then diluted with EtOAc (40
mL), washed with H2O (30 mL), 5% aq NaHCO3 (30 mL) and
H2O (30 mL), dried over Na2SO4, filtered, and evaporated to
dryness under reduced pressure. The residue was purified
by silica gel column chromatography (20-60% EtOAc in
petroleum ether, v/v) to afford nucleoside 3a as a yellowish
solid material (118 mg; 74%). Rf ) 0.60 (60% EtOAc in
petroleum ether, v/v); ESI-HRMS m/z 1106.4259 ([M+Na]+,
C66H62N5O8P·Na+ calc. 1106.4227); 31P NMR (CDCl3) δ 149.1.
a Thermal denaturation temperatures [Tm values (°C) (∆Tm ) change
in Tm value calculated relative to DNA/DNA or DNA/RNA reference
duplex)] measured as the maximum of the first derivative of the melting
curve (A260 vs temperature) recorded in 10 mM phosphate buffer ([Na+]
) 110 mM, [Cl-] ) 100 mM, [EDTA] ) 0.1 mM, pH 7.0) using 1.0
µM concentrations of the two complementary strands. Tm values are
averages within (1 °C of at least two measurements. See Scheme 1 for
structure of monomer X and monomer Y. MALDI-MS m/z ([M-H]-;
found/calc.): ON3, 3092/3093; ON4, 3020/3022; ON5, 3021/3022;
ON6, 3360/3361; ON7, 3161/3163; ON8, 3088/3092; ON9, 3090/3092;
ON10, 3500/3501. b ON1 is the cDNA strand to ON2/ON4/ON5/ON6/
ON8/ON9/ON10; ON2 is the cDNA strand to ON3/ON7. c Tm values
(°C) toward DNA strands containing a single mismatch in the central
position, monomer X: G/C/T: 24.0/31.5/28.0; monomer Y: G/C/T: 36.5/
30.0/35.5. d Tm values (°C) toward RNA strands containing a single
mismatch in the central position, monomer X: G/C/U: 21.0/21.0/18.5;
monomer Y: G/C/U: 30.0/17.0/22.0.
Thermal Denaturation Studies. Concentrations of oligo-
nucleotides were calculated using the following extinction
coefficients (OD/µmol): G, 10.5; A, 13.9; T/U, 7.9; C, 6.6;
coronene units, 36.0. The two complementary strands (∼1.0 µM)
were thoroughly mixed, and the duplex denatured by heating
to 80 °C followed by cooling to the starting temperature (5 °C)
of the experiment. Quartz optical cells with a path length of
1.0 cm were used. Thermal denaturation curves were obtained
on a Perkin-Elmer Lambda 35 UV/vis spectrometer equipped
with a PTP-6 peltier temperature programmer. Melting tem-
peratures (Tm values/°C) determined as the maximum of the
first derivative of the thermal denaturation curve (A260 vs
temperature) recorded in 10 mM phosphate buffer ([Na+] )
110 mM, [Cl-] ) 100 mM, [EDTA] ) 0.1 mM, pH 7.0). A
temperature range from 5 to 75 °C with a ramp of 1.0 °C/min
was used.
(1R,3R,4R,7S)-5-[4-(Coronen-1-yl)-4-oxo-butanoyl]-1-(4,4′-
dimethoxytrityloxy methyl)-7-hydroxy-3-(thymin-1-yl)-2-
oxa-5-azabicyclo[2.2.1]heptane (2b). Nucleoside 1 (58) (0.40
g, 0.69 mmol) was dissolved in anhydrous dichloromethane (20
mL). To this stirred solution were added N-(3-dimethylamino-
propyl-N′-ethylcarbodiimide hydrochloride (EDC ·HCl, 0.14 g,
0.76 mmol) and 3-coronenyl propionic acid (0.30 g, 0.76 mmol)
at 0 °C. After warming to rt, the reaction mixture was stirred
for 18 h, whereupon it was diluted with dichloromethane (50
mL) and successively washed with brine (40 mL) and H2O (40
mL). The organic phase was evaporated to dryness under
reduced pressure, and the resulting residue was purified by silica
gel column chromatography (0-2% MeOH in CH2Cl2, v/v) to
1
afford a rotameric mixture (∼1:0.43 by H NMR) of 2b as a
yellowish solid material (380 mg; 58%). Rf ) 0.32 (7% MeOH
in CH2Cl2, v/v); ESI-HRMS m/z 976.3291 ([M+Na]+,
C60H47N3O9 ·Na+ calc. 976.3210); 1H NMR (DMSO-d6) δ
UV Absorption Measurements. UV absorption measure-
ments (200-500 nm) were performed on a Shimadzu UV-160A

Coronene Functionalized 2′-Amino-LNA
Bioconjugate Chem., Vol. 21, No. 3, 2010 515
Table 2. Thermal Denaturation Studies in Zipper Arrangementsa
(selected signals) 11.50 (br s, NHA), 11.35 (br s, NHB),
9.53-8.92 (m, Ar), 7.79 (s, H6), 7.63-6.82 (m, Ar), 5.68 (s,
H-1′A), 5.52 (br s, H-1′B), 4.77 (br s, H-2′B), 4.63 (br s, H-2′A),
4.14 (br s, H-3′A and H-3′B), 1.83 (br s, CH3-A), 1.16 (br s,
CH3-B); 13C NMR (DMSO-d6) δ 203.6, 202.1, 170.7, 170.5,
164.0, 157.8, 150.1, 148.3, 140.2, 134.9, 134.9, 133.9, 132.1,
132.0, 129.2, 129.1, 128.9, 128.4, 128.3, 128.0, 127.9, 127.6,
127.4, 127.1, 126.9, 126.8, 126.6, 126.5, 126.4, 126.2, 126.0,
124.8, 124.0, 122.5, 121.9, 121.2, 121.1, 120.9, 120.7, 113.9,
112.7, 109.9, 108.3, 108.2, 89.4, 88.8, 86.4, 79.9, 68.4, 63.3,
56.6, 55.0, 54.9, 50.8, 45.6, 42.0, 36.9, 28.5, 12.4, 10.9.
(1R,3R,4R,7S)-5-[4-(Coronen-1-yl)-4-oxo-butanoyl]-7-[2-
cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimeth-
oxytrityloxymethyl)-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]-
heptane (3b). Nucleoside 2b (0. 32 g, 0.33 mmol) was dissolved
in a mixture of anhydrous acetonitrile (7.0 mL) and dichlo-
romethane (2.0 mL) under stirring at rt. N,N-Diisopropylam-
monium tetrazolide (0.057 g, 0.50 mmol) and bis(N,N-
diisopropylamino)-2-cyanoethoxyphosphine (0.31 mL, 1.00
mmol) were added under nitrogen. The reaction mixture was
stirred for 2 h and then diluted with EtOAc (60 mL), washed
successively with H2O (40 mL), 5% aq NaHCO3 (40 mL) and
H2O (40 mL), dried over Na2SO4, filtered, and evaporated to
dryness under reduced pressure. The residue was purified by
silica gel column chromatography (30-90% EtOAc in petro-
leum ether, v/v) to afford nucleoside 3b as a yellowish solid
material (300 mg; 79%). Rf ) 0.60 (80% EtOAc in petroleum
ether, v/v);ESI-HRMSm/z1176.4282([M+Na]+,C69H64N5O10P·
Na+ calc. 1176.4333); 31P NMR (CDCl3) δ 149.0, 150.2.
a Thermal denaturation temperatures were recorded under the same
conditions as described in Table 1. See Scheme 1 for structures of
monomer X and monomer Y. The black droplets illustrate the coronene
moieties of X and Y monomers.
RESULTS
The bicyclic nucleoside 1 was synthesized according to a
published procedure (58) and was selectively N-alkylated by
reductive amination using coronene-1-carbaldehyde producing
N-(coronen-1-yl)methyl derivative 2a. Furthermore, 1 was
N-acylated by 4-(coronen-1yl)-4-oxobutanoic acid giving the
corresponding nucleoside 2b. The phosphoramidite building
blocks 3a and 3b were obtained by standard phoshphitylation
of nucleosides 2a and 2b, respectively, using bis(N,N-diisopro-
pylamino)-2-cyanoethoxyphosphine as phosphitylating agent
(Scheme 1).
Synthesis of oligonucleotides (ONs) containing 2′-amino-
LNA monomers X and Y was performed in 0.2 µmol scale using
an automated DNA synthesizer. 1H-Tetrazole was used as
coupling agent for monomers X and Y resulting in 95% and
99% stepwise coupling yields, respectively, based on the
absorbance of the dimethoxytrityl cation released after each
coupling step (see Experimental Procedures section for details).
All synthesized ONs were purified by RP-HPLC, and their
identity was confirmed by MALDI-TOF mass spectroscopy and
ion-exchange HPLC, respectively.
The influence of the functionalized 2′-amino-LNA monomers
X and Y on duplex thermal stabilities was studied toward DNA
and RNA complements (Table 1) by UV thermal denaturation
experiments using a 10 mM phosphate buffer ([Na+] ) 110
mM, [Cl-] ) 100 mM, [EDTA] ) 0.1 mM, pH 7.0). In all
cases, the denaturation curves displayed monophasic transitions.
Earlier, it was reported that conjugates containing function-
alized 2′-amino-LNA monomers form stable duplexes toward
both DNA and RNA complements, and that N2′-acylated 2′-
amino-LNA derivatives exhibit higher thermal stabilities than
the corresponding N2′-alkylated derivatives (18, 49). Our results
are in good agreement with these earlier results. Incorporation
of a single monomer X in mixed 9-mer ON3 and ON5 induced
an increase in duplex stability toward cDNA (∆Tm ) +2.0 and
+3.0 °C compared to the unmodified reference duplex ON1:
ON2) (Table 1). A similar effect was observed for ON3 and
Figure 1. Steady-state fluorescence emission spectra of single-stranded
probe ON3 (with monomer X) and of duplexes formed between ON3
and cDNA (ON3:DNA) or RNA (ON3:RNA).
ON5 with complementary RNA. Surprisingly, ON4 showed
minor destabilization toward cDNA (∆Tm ) -1.5 °C) and
complementary RNA (∆Tm ) -0.5 °C). In contrast to the
melting temperature results of monomer X, a single incorpora-
tion of monomer Y in mixed DNA 9-mer ONs (ON7-ON9)
induced high-affinity hybridization toward DNA (∆Tm ) +9.0
to +15.0 °C) and RNA (∆Tm ) +9.5 to +12.0 °C). When
comparing thermal stabilities of duplexes involving ONs singly
functionalized with monomer Y, (ON7-ON9) were higher than
those of the corresponding unmodified duplexes and of duplexes
involving a perylenemethyl-2′-amino-LNA monomer (51), (phe-
nylethynyl)pyrene-functionalized-2′-amino-LNAs (53) and N2′-
pyrenecarbonyl-functionalized 2′-amino-LNAs (18). This indi-
cates that attachment of coronene moieties to 2′-amino-LNA
monomers is compatible with duplex formation. To evaluate
this further, we performed CD experiments on duplexes formed
between ON8 or ON9, each containing one Y monomer, and
cDNA or RNA (S7, ESI). These spectra indicate similar duplex
structures of the modified duplexes and the unmodified reference
(ON2:DNA and ON2:RNA) duplexes, which support that one
Y modification can be accommodated without significantly
disturbing overall duplex structure.
Incorporation of two modifications may, however, have a
more profound effect on duplex structure. N2′-Substituents of
2′-amino-LNAsareknowntopointtowardtheminorgroove(15,49)
and may therefore be affected by the narrow minor grooves in
A-type duplexes (59). Incorporation of two X monomers
separated by one nucleotide in a 9-mer DNA strand (ON6)

516 Bioconjugate Chem., Vol. 21, No. 3, 2010
Gupta et al.
Figure 2. Steady-state fluorescence emission spectra of single-stranded
probe ON7 (with monomer Y) and of duplexes formed between ON7
and cDNA (ON7:DNA) and RNA (ON7:RNA).
Figure 4. Steady-state fluorescence emission spectra of single-stranded
probe ON7 (with monomer X) and of duplexes formed between ON7
and ON8 (“-1 zipper” constitution) and ON7 and ON9 (“+1 zipper”
constitution).
satisfactory in both DNA and RNA contexts: T:T/U (-7.5 °C/
-14.0 °C, respectively), T:G (-6.5 °C/-6.0 °C, respectively),
and T:C (-13.0 °C/-19.0 °C, respectively).
Studies with X/Y monomers positioned in both strands of a
duplex were performed in -1 and +1 interstrand zipper (15)
arrangements of monomer X or Y (Table 2). Insertion of two
X monomers in a “-1 zipper” constitution induced extensively
increased thermal stability (Table 2, ∆Tm ) +20.5 °C), as
did insertion of two Y monomers (+28.5 °C). On the other
hand, insertion of two X monomers in a “+1 zipper”
constitution induced a limited increase in thermal stability
(+5.0 °C), whereas insertion of two Y monomers in a “+1
zipper” constitution again induced a significant increase in
thermal stability (+18.5 °C). The thermal denaturation temper-
atures of duplexes involving two modified strands are thus
remarkably higher than those of duplexes containing monomers
X or Y in only one strand, indicating that interstrand com-
munication between coronene moieties in the studied “zipper”
pattern contributes to overall duplex stability.
Figure 3. Steady-state fluorescence emission spectra of single-stranded
probe ON3 (with monomer X) and of duplexes formed between ON3
and ON4 (“-1 zipper” constitution) and ON3 and ON5 (“+1 zipper”
constitution).
The steady-state fluorescence emission spectra of ON3-ON6
containing monomer X when excited at its UV absorption
maximum λmax 310 nm (10 °C, (1 °C) showed three emission
bands. Insertion of a single X monomer in ONs (ON3-ON5)
in general results in quenching of fluorescence intensities (up
to half of the intensity of single-stranded ONs) upon hybridiza-
tion to cDNA or RNA, though a minor increase of fluorescence
intensity in the case of ON4 was observed upon hybridization
to cDNA. (Figure 1, and ESI S4.1 to S4.4). However, ON6
with two X monomers separated by one nucleotide upon
hybridization with cDNA displayed increased fluorescence
intensity (∼2-fold) with unchanged emission wavelength and
upon hybridization with complementary RNA slightly increased
fluorescence intensity at nearly the same emission wavelength
(see ESI S4.5). Incorporation of monomer X in ONs
(ON3-ON6) results in fluorescence emission maxima at ∼429
nm, which is 25 nm red-shifted relative to the corresponding
pyrenecarbonyl-2′-amino-LNA ONs (18), 61 nm blue-shifted
relative to the corresponding perylene-3-carbonyl-2′-amino-LNA
ONs (52) and 1-26 nm blue-shifted relative to the correspond-
ing (phenylethynyl)pyrenecarbonyl-2′-amino-LNA ONs (53).
In contrast, insertion of a single Y monomer into ONs
(ON7-ON9) in general results in a marked red shift with similar
or increased fluorescence intensities upon hybridization to cDNA
or RNA (Figure 2 and ESI, S4.6 to S4.9). Surprisingly, ON10
containing two Y monomers upon duplex formation with DNA/
resulted in two melting transitions, which we at present are
unable to explain; no transition was observed for ON6 without
a complementary strand. On the contrary, single transitions upon
incorporation of two Y monomers separated by one nucleotide
(ON10) were obtained with enhanced thermal stability of the
duplex with cDNA (∆Tm ) +1.5 °C per modification) but
decreased thermal stability of the duplex with complementary
RNA (∆Tm ) -2.7 °C per modification). Relative destabiliza-
tion toward DNA/RNA complements upon sequential incorpo-
ration of two monomers Y (relative to incorporation of one Y
monomer) suggests that the minor groove is too narrow to
accommodate the two coronene moieties.
The Watson-Crick base pair selectivity of monomers X and
Y was evaluated by observing thermal denaturation temperatures
of duplexes formed between ON3/ON7 and DNA or RNA
strands containing a single mismatched nucleotide opposite
monomer X or Y (see Table 1; footnotes c and d). The base
pair selectivity of monomer X was satisfactory in the case of
T:G DNA and T:U RNA mismatches (-7.5 °C/-9.5 °C,
respectively). A relatively weak decrease in thermal stability
was observed for the T:T DNA mismatch (-3.5 °C), whereas
no mismatch discrimination was observed for the T:C DNA
mismatch. Against RNA, ON3 showed appreciable mismatch
discrimination. The base pairing selectivity of monomer Y was

Coronene Functionalized 2′-Amino-LNA
Bioconjugate Chem., Vol. 21, No. 3, 2010 517
Figure 5. Energy-minimized structures of duplexes. (5a) ON3:DNA (monomer X in side view); (5b) ON3:DNA (monomer X in top view); (5c)
ON7:DNA (monomer Y in side view); (5d) ON7:DNA (monomer Y in top view); (5e) ON3:ON4 (monomer X, “-1 zipper” arrangement); (5f)
ON3:ON5 (monomer X, “+1 zipper” arrangement); (5g) ON7:ON8 (monomer Y, “-1 zipper” arrangement); and (5h) ON7:ON9 (monomer Y,
“+1 zipper” arrangement) in side view. Color code: Sugar phosphate backbone (red); coronene (blue); nucleobases (green).
RNA complements showed a slight shift in emission maxima
(∼4 nm) to shorter wavelengths with a weakly increased
fluorescence intensity (∼1.5-fold) (see ESI S4.10).
(recorded at 10 °C) are shown in the electronic Supporting
Information (Figures S4.1, S4.2, S4.6, and S4.7). Probe ON3
was seen to be equally sensitive to the presence of mismatched
nucleosides in DNA and RNA targets with a ∼2-fold increase
in fluorescence intensity for all singly mismatched complexes
(Supporting Information Figures S4.1-S4.2). For probe ON7
containing a single monomer Y, the fluorescence effect of
mismatches relative to the single-stranded ON7 and fully
matched duplexes did not allow straightforward detection of
mismatches (Supporting Information Figures S4.6-S4.7).
All duplexes having monomers X in both complementary
strands displayed an unstructured emission band at λem ∼ 430
nm. The fluorescence intensity of the “zippers” was lower in
“-1 zipper” constitution and slightly higher in “+1 zipper”
constitution than of the previously described duplexes having
coronene monomer X in only one strand (Figure 3). Duplexes
Incorporation of a single Y monomer in ONs (ON7-ON10)
thus results in a broad, unstructured band with fluorescence
emission maxima at ∼453 nm, which is 51 nm red-shifted
relative to pyrenecarbonyl-2′-amino-LNA ONs (18), 37 nm blue-
shifted relative to perylene-3-carbonyl-2′-amino-LNA ONs (52),
and 7-23 nm blue-shifted relative to (phenylethynyl)pyrene-
carbonyl-2′-amino-LNA ONs (53). No characteristic changes
in the shape of the spectral curves were observed.
Next, fluorescence properties of duplexes formed by ON3/
ON7 and singly mismatched DNA/RNA targets were investi-
gated. Noteworthy, as mentioned earlier, the mismatched
duplexes display Tm values of more than 19 and 30 °C for ON3
and ON7, respectively. The resulting fluorescence spectra

518 Bioconjugate Chem., Vol. 21, No. 3, 2010
Gupta et al.
having monomer Y in both complementary strands displayed
an unstructured emission band at λem ∼ 484 nm. In contrast,
the fluorescence intensity of the “zippers” was higher in “-1
zipper” constitution and lower in “+1 zipper” constitution than
of the previously described duplexes having coronene monomer
Y in only one strand (Figure 4). Excimer emission was observed
neither in single-stranded ONs (ON3-ON10) nor in their
duplexes with DNA/RNA complements, whereas microcrystals
of coronene showed both monomer and excimer emission bands
(60). In contrast, insertion of pyrene-functionalized LNAs in
“-1 zipper” motif resulted in strong excimer formation com-
bined with increased thermal stability (15, 53).
Molecular modeling of duplexes formed between ON3
(containing monomer X) or ON7 (containing monomer Y) and
cDNA (ON2) was performed using the AMBER force
field (61, 62) as implemented in MacroModel V 8.5 (63).
Interstrand zipper arrangement modeling were also performed
using the same method. All 9-mer duplexes were constructed
with the coronene moiety of monomers X and Y in an
extrahelical position. The observed low-energy structures were
used for molecular dynamics simulations.
The model structure of the ON3:ON2 hybrid suggests that
the attachment of the methylcoronene moiety at the 2′-nitrogen
atom of 2′-amino-LNA does not induce significant changes in
the duplex geometry and that coronene binds in the minor
groove without stacking with the nucleobases (Figure 5a,b). On
the other hand, the model structure of hybrid ON7:ON2 revealed
many low-energy conformations during dynamics. This can be
explained by the more flexible nature of the 4-oxobutanoyl linker
of monomer Y though the coronene moiety shown still to be
positioned in the minor groove (Figure 5c,d). Modeling results
of zipper hybrid ON3:ON4 showed the two coronene moieties
to be stacking while being positioned in the minor groove
(Figure 5e), which could explain the high Tm value measured
for this “-1 zipper” duplex. This is substantiated for the ON3:
ON5 hybrid in which the two coronene moieties only partially
stack (Figure 5f), thus offering an explanation of the lower Tm
value observed in this case. The model structure of hybrid ON7:
ON8 showed the closest arrangement of the two coronene
moieties stacking with each other and with nucleobases (Figure
5g), which correlates with the very high Tm value measured.
Hybrid ON7:ON9 showed the two coronene moieties to nicely
stack in the minor groove (Figure 5h), but without stacking with
the nucleobases.
properties of the coronene-modified ONs introduced herein, such
as fluorescence induced mismatch discrimination and a batho-
chromic shift in UV absorption upon hybridization with cDNA/
RNA, testify to a significant potential for applications in nucleic
acid based diagnostics.
ACKNOWLEDGMENT
The Nucleic Acid Center is a research center of excellence
funded by the Danish National Research Foundation. We thank
the Danish National Research Foundation for financial support.
Note Added after ASAP Publication: Due to a production
error, an uncorrected version of this paper was inadvertently
published on Jan 25, 2010. The corrected version was reposted
on Feb 3, 2010.
Supporting Information Available: Steady-state fluorescence
studies of ONs; protocol for molecular modeling studies; absorption
and emission maxima of ONs; steady-state fluorescence emission
spectras; An illustrative example of thermal denaturation curve and
1H NMR spectra of 2a and 2b. This material is available free of
charge via the Internet at http://pubs.acs.org.
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