Fluorescent oligonucleotides containing a novel perylene 2'-amino-α-L-LNA monomer: Synthesis and analytical potential

Article abstract of DOI:10.1135/cccc2011096

Herein, a novel fluorescent nucleotide analogue, perylene-2'-amino-α- L-LNA, has been prepared and studied within synthetic oligonucleotides of different sequences. The phosphoramidite reagent was synthesized in 85% overall yield starting from 2'-amino-a-

Full text of DOI:10.1135/cccc2011096

Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
1347
FLUORESCENT OLIGONUCLEOTIDES CONTAINING A NOVEL
PERYLENE 2′-AMINO-α-L-LNA MONOMER: SYNTHESIS AND
ANALYTICAL POTENTIAL
a,b
a2
Irina V. ASTAKHOVAa1,*, T. Santhosh KUMAR and Jesper WENGEL
a
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark,
1
2
DK-5230 Odense M, Denmark; e-mail: ias@ifk.sdu.dk, jwe@ifk.sdu.dk
Present address: Chemical Biology Unit, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bldg. 8, Rm. 121, NIH, NIDDK, LCBB, Bethesda,
MD 20892-0810, USA; e-mail: thatikondas@niddk.nih.gov
b
Received May 4, 2011
Accepted June 10, 2011
Published online November 20, 2011
Dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition of his
outstanding contributions to the area of nucleic acid chemistry.
Herein, a novel fluorescent nucleotide analogue, perylene-2′-amino-α-L-LNA, has been pre-
pared and studied within synthetic oligonucleotides of different sequences. The
phosphoramidite reagent was synthesized in 85% overall yield starting from 2′-amino-
α-L-LNA nucleoside. Incorporation efficiency of the resulting perylene-2′-amino-α-L-LNA
monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of
the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligo-
nucleotides containing monomer T* showed high binding affinity towards complementary
DNA and RNA targets, batochromically shifted excitation/emission wavelengths with respect
to the often applied polyaromatic hydrocarbon pyrene, high fluorescent quantum yields and
very low target detection limits (5–10 nM). Fluorescence of single stranded LNA/DNA
mixmer oligonucleotide having two incorporations of monomers T* was quenched (quan-
tum yield ΦF = 0.21) relative to duplexes of this probe with complementary DNA and RNA
(ΦF = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon
target binding was demonstrated by two short oligonucleotides of analogues sequences con-
taining monomers T* at 5′- and 3′-terminal positions. We explain the hybridization-induced
light-up effect observed for double-labeled probe by a reduction of fluorescence quenching
due to precise positioning of the fluorophores within the double-stranded complexes. Fur-
thermore, we propose that a covalent link between two T* monomers in the double-labeled
probe provides a remarkable degree of rigidity in the double helix which enforces position-
ing of the bulky perylene moieties in the nonpolar groove resulting in reduced fluorescence
quenching.
Keywords: DNA; Fluorescence; Fluorescent probes; Oligonucleotides; Nucleosides; Nucleo-
tides; LNA; Molecular diagnostics.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1347–1360
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011096

1348
Astakhova, Kumar, Wengel:
Detection of nucleic acids is an extremely important task both in research
and industry. This includes in applications relating to diagnostics of genetic
and infectious diseases1, antisense technology2, fluorescent imaging3 and
other biomedical studies. After remarkable progress in automatic oligo-
nucleotide synthesis achieved in the last three decades, fluorescently la-
beled oligonucleotides have become readily available as useful tools for
nucleic acid detection and analysis. In order to be an effective nucleic acid
diagnostic probe, the labeled oligonucleotide has to display high affinity
and selectivity of target binding and considerable change of fluorescence
upon formation of complexes with complementary strands4. Continuous
development of such oligonucleotide probes has led to several formats of
fluorescence nucleic acid assays, e.g. detection by fluorescence polarization5
or fluorescence resonance energy transfer (FRET)6, Taqman probes (also
known as 5′-nuclease probes)7, sunrise primers8, molecular beacons9, du-
plex scorpion primers10, and oligonucleotides labeled with a single kind of
a hybridization-sensitive substance11–17. Each of these methods and con-
structs has its own specific strengths and weaknesses, but there is still no
universal approach of choice18. Moreover, in order to simultaneously ana-
lyze multiple targets it is desirable to have various fluorescent dyes display-
ing different excitation/emission wavelengths19.
Among polyaromatic hydrocarbons, pyrene has long been the most pop-
ular dye for nucleic acid labeling (Fig. 1). However another polyaromatic
molecule, perylene, is currently an object of increasing interest as a highly
sensitive dye to measure local microenvironment changes within nucleic
acids, e.g. those caused by hybridization20–22. Fluorescence of perylene is
typically observed at 450–500 nm and its excited lifetime is relatively short
(4–5 ns) while the fluorescence quantum yield is very high (0.5–1.0)23. Fur-
thermore, perylene displays a stronger fluorescence anisotropy effect than
pyrene24. High fluorescence quantum yield, red-shifted emission compared
to pyrene (350–500 nm and 420–620 nm for pyrene and perylene in cyclo-
hexane, respectively23), and the remarkable photostability of perylene
makes it an attractive label for fluorescence-based detection both in vitro
and in vivo. We previously introduced 2′-N-(perylen-3-yl)carbonyl-
2′-amino-LNA modification which induced high thermal stability of
DNA:DNA and DNA:RNA duplexes, high Watson–Crick mismatch selectiv-
ity, red shifted fluorescence emission compared to pyrene, and high fluo-
rescence quantum yields25. Additionally, the perylene-LNA probe was
successfully applied for detection of mRNA in vivo providing an excitation
wavelength which completely eliminated cell autofluorescence25.
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Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
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Besides insertion of fluorescent dyes, chemical modification of oligo-
nucleotides allows improvement of their binding affinity and selectivity to-
wards complementary DNA and RNA targets. These properties have in
particular been reported for N-functionalized 2′-amino locked nucleic acids
(2′-amino-LNA 26 and isomeric 2′-amino-α-L-LNA 27), also containing
FIG. 1
Chemical structures of the fluorescent polyaromatic hydrocarbons pyrene and perylene, which
are often used for oligonucleotide labeling, and of N-functionalized fluorescent locked nucleic
acid analogues. R = polyaromatic hydrocarbon residue, also indicated with rays
a
b
c
FIG. 2
Representative structures of the perylene labeled oligonucleotides prepared in this study and
their hybridization with DNA/RNA targets: (a) 12mer single-labeled probes, (b) 17mer double-
labeled probes and (c) 8mer+9mer dual-probes. Black droplets indicate monomer T*
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Astakhova, Kumar, Wengel:
polyaromatic hydrocarbons (Fig. 1). Stimulated by recently described high
affinity and selectivity of hybridization provided by 2′-amino-α-L-LNA
derivatives27, sensitivity of pyrene-functionalized 2′-amino-α-L-LNA and
perylene-functionalized 2′-amino-LNA probes to hybridization25,28, and tak-
ing the high fluorescence quantum yields and red-shifted fluorescence
emission of perylene into account, we have prepared a novel perylene-
modified 2′-amino-α-L-LNA derivative. In this paper, we describe synthesis
of 2′-N-(perylen-3-yl)methyl-2′-amino-α-L-LNA monomer T* and its incor-
poration into synthetic oligonucleotides, as well as thermal denaturation
studies and spectral properties of the resulting oligonucleotide probes of
different sequences containing either single or double insertions of mono-
mer T* (Fig. 2).
EXPERIMENTAL
Synthesis of 2′-N-(Perylen-3-yl)methyl-2′-amino-α-L-LNA Phosphoramidite Reagent
Reagents obtained from commercial suppliers were used as received; 3-perylenecarb-
aldehyde29 and diisopropylammonium tetrazolide30 were synthesized as described. Perylene
used as standard for fluorescence quantum yield measurements was recrystallized. HPLC
grade MeOH, EtOAc and petroleum ether were stored over activated 4Å molecular sieves.
DCM was always used freshly distilled over CaH2. Other solvents were used as received. Pho-
tochemical studies were performed using spectroquality cyclohexane. NMR spectra were re-
corded at 303 K on a Varian Gemini 2000 300 MHz instrument. Chemical shifts (δ-scale) are
reported in ppm relative to solvents peaks (DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for
13C; 85% aq. H3PO4: 0.00 ppm for 31P). 1H NMR coupling constants (J) are reported in Hz
and refer to apparent multiplicities. High resolution ESI-mass spectra were recorded in posi-
tive ion mode using a PE SCIEX QSTAR pulsar mass spectrometer. Analytical thin-layer chro-
matography was performed on Kieselgel 60 F254 precoated aluminium plates (Merck). Silica
gel column chromatography was performed using Merck Kieselgel 60 0.040–0.063 mm.
(1S,3R,4S,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-5-(perylen-3-yl)methyl-
3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (2)
Nucleoside 1 31 (200 mg, 0.35 mmol) was dried by coevaporation with anhydrous DCM (2 ×
10 ml) and dissolved in anhydrous 1,2-dichloroethane (5 ml). To this solution, 3-perylene-
carbaldehyde29 (145 mg, 0.54 mmol) and NaBH(OAc)3 (112 mg, 0.54 mmol) were subse-
quently added, and the reaction mixture was stirred at r.t. for 12 h. Absolute EtOH–
toluene–Et3N mixture (9:9:2, v/v/v) was then added, and the resulting material was evapo-
rated to dryness under reduced pressure. The residue obtained was purified by silica gel col-
umn chromatography (0–5% MeOH in DCM, v/v) to afford the N-alkylated product 2
(264 mg, 90%) as a white solid material (a mixture of rotamers according to NMR). RF 0.47
(5% MeOH, 1% NEt3, DCM, v/v/v). 1H NMR (300 MHz, DMSO-d6; the signals are given for
the major rotamer): 11.16 (s, 1 H, NH); 8.37–6.85 (m, 26 H, Ar); 6.00 (d, 1 H, J = 3.6, 3′-OH);
5.90 (d, 1 H, J = 1.8, H-1′); 4.45 (d, 1 H, J = 3.6, H-3′); 4.40 (d, 1 H, J = 13.2, CH2Ar); 4.20 (d,
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Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
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1 H, J = 13.1, CH2Ar); 3.72 (s, 6 H, 2 × CH3O); 3.47 (s, 1 H, H-2′); 3.35 (d, 1 H, J = 10.6,
H-5′a); 3.30 (d, 1 H, J = 10.6, H-5′b); 3.25 (d, 1 H, J = 9.8, H-5′′a); 3.00 (d, 1 H, J = 9.8,
H-5′′b); 1.63 (s, 3 H, CH3). 13C NMR (300 MHz, DMSO-d6): 163.8, 158.0, 150.1 (Ar), 144.7
(Ar), 136.6 (Ar), 135.4 (Ar), 135.3 (Ar), 134.1 (Ar), 132.6 (Ar), 130.5 (Ar), 130.4 (Ar), 130.3
(Ar), 129.8 (Ar), 129.6 (Ar), 128.1 (Ar), 127.8 (Ar), 127.6 (Ar), 126.8 (Ar), 126.6 (Ar), 126.0
(Ar), 124.3 (Ar), 123.7 (Ar), 120.5 (Ar), 120.3 (Ar), 119.7 (Ar), 113.1 (Ar), 106.9, 90.4, 85.3
(C-1′), 85.1, 74.9 (C-3′), 65.4 (C-2′), 60.9 (C-5′), 58.8 (C-5′′), 58.6 (CH2Ar), 54.9 (CH3O), 54.8
(CH3O), 12.3 (CH3). ESI-HRMS: m/z 836.3342 ([M + Na]+, C53H45N3O7Na+ calculated
836.3330).
(1S,3R,4S,7R)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-
1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-5-(perylen-3-yl)methyl-
3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (3)
The LNA nucleoside derivative 2 (150 mg, 0.18 mmol) was coevaporated with anhydrous
DCM (2 × 5 ml) and dissolved in anhydrous DCM (5 ml). To this solution, diisopropyl-
ammonium tetrazolide (62 mg, 0.36 mmol) and bis(N,N-diisopropylamino)-2-cyanoethoxy-
phosphine (115 µl, 0.36 mmol) were added under argon, and the resulting mixture was
stirred at r.t. for 12 h. The mixture was diluted with DCM (50 ml) and washed consecutively
with a saturated solution of NaHCO3 (2 × 50 ml) and brine (50 ml). The organic layer was
dried over Na2SO4, evaporated to dryness under reduced pressure and the residue was puri-
fied by column chromatography on silica gel (30–50% EtOAc in petroleum ether, containing
1% NEt3, v/v/v) to give phosphoramidite 3 as a white solid material. Yield 176 mg (94%).
RF 0.45, 0.43 (50% EtOAc, 1% NEt3, petroleum ether, v/v/v). 31P NMR (300 MHz, DMSO-d6):
149.9, 146.6 (1:1.7) (main diastereomer). ESI-HRMS: m/z 1058.4250 ([M
C
+
Na]+,
62H62N5O8PNa+ calculated 1058.4228).
Synthesis and Purification of Modified Oligonucleotides
LNA phosphoramidites were obtained from commercial supplier (Exiqon). Oligonucleotide
synthesis was carried out on a PerSpective Biosystems Expedite 8909 instrument in 200 nmol
scale using standard manufacturer’s protocols, except for increased coupling time for modi-
fied phosphoramidite 3 and LNA 32. 3′-Labeled oligonucleotide ON6 was prepared on CPG
universal support III (Glen Research) using manufacturer’s protocol. In case of 3, 30 min
coupling time using 0.6 M pyridine hydrochloride in MeCN as an activator was applied,
while for LNA phosphoramidites 10 min coupling time and 1H-tetrazole solution in MeCN
were applied. Coupling yields based on the absorbance of the dimethoxytrityl cation re-
leased after each coupling were approximately 95–99% for modified phosphoramidite 3,
98–99% for LNA, and 99–100% for unmodified DNA phosphoramidites. Cleavage from solid
support and removal of nucleobase protecting groups were performed using standard condi-
tions (32% aqueous ammonia at 55 °C for 12 h). In case of ON6 cleavage from the universal
solid support was performed using 3.5 M solution of ammonia in methanol (1 h) followed
by removal of nucleobase protecting groups with 32% aqueous ammonia at r.t. for 12 h.
Unmodified DNA/RNA strands were obtained from commercial suppliers and used without
further purification, while all the modified oligonucleotides were purified by DMT-ON
RP-HPLC using the Waters Prep LC 4000 equipped with Xterra MS C18-column (10 µm,
300 mm × 7.8 mm). Elution was performed starting with an isocratic hold of A-buffer for
5 min followed by a linear gradient to 55% B-buffer at a flow rate of 1.0 ml/min over
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Astakhova, Kumar, Wengel:
75 min (A-buffer: 95% 0.1 M NH4HCO3, 5% CH3CN; B-buffer: 25% 0.1 M NH4HCO3,
75% CH3CN). RP purification was followed by detritylation (80% aqueous AcOH, 30 min),
precipitation (acetone, –18 °C, 12 h) and washing with acetone twice. The identity and pu-
rity of oligonucleotides were verified by MALDI-TOF mass spectrometry (recorded on an
Applied Biosystems Voyager-DE STR spectrometer) and ion-exchange HPLC (purities > 90%),
respectively. Measured masses of the oligonucleotides (calculated masses m/z for [M – H]–):
ON1: 3730 (3733); ON2: 3728 (3733); ON3: 3732 (3733); ON5: 5305 (5309); ON6: 2455
(2455), ON7: 2790 (2792). For sequences of oligonucleotides prepared in this study, see
Tables I, II.
UV-Visible Absorption and Thermal Denaturation Studies
UV-Visible absorption and thermal denaturation studies were performed on a Perkin–Elmer
Lambda 35 UV/VIS Spectrometer equipped with PTP 6 (Peltier Temperature Programmer) in
a medium of salt phosphate buffer (100 mM sodium chloride, 10 mM sodium phosphate,
0.1 mM EDTA, pH 7.0). Concentrations of oligonucleotides were calculated using the follow-
ing extinction coefficients (OD260/µmol): G, 10.5; A, 13.9; T/U, 7.9; C, 6.6; T*, 33.2. Mod-
ified oligonucleotides and target strands (0.5–1.0 µM each strand) were thoroughly mixed,
denaturated by heating and subsequently cooled to the starting temperature of experiment.
Thermal denaturation temperatures (Tm values, °C) were determined as the maxima of the
first derivative of the thermal denaturation curves (A260 vs temperature). Reported Tm values
are an average of two measurements within 1.0 °C.
Fluorescence Steady-State Emission Studies, Quantum Yield and
Limits of Target Detection Determinations
Fluorescence spectra were obtained at 19 °C in a medium of salt phosphate buffer (see
above) using a Perkin–Elmer LS 55 luminescence spectrometer and an excitation wavelength
of 425 nm, excitation slit of 4.0 nm, emission slit of 2.5 nm, scan speed of 120 nm/min,
and 0.1–0.5 µM concentrations of the single-stranded probes or the corresponding duplexes.
The fluorescence quantum yields (Φf) were measured by the relative method using perylene
(Φf = 0.93) in cyclohexane as a standard33. The optical density of the solutions in a 1-cm
quartz cuvette at the excitation wavelength did not exceed 0.1. The values of Φf were
corrected with the refractive index of the solvent; the measured refractive indexes of cyclo-
hexane and phosphate buffer were 1.426 and 1.334 at 19 °C, respectively. For target titra-
tion fluorescence measurement in limit of target detection (LOD) determinations34, the
relevant fluorescent probe (500 nM each strand) was mixed with a series of solutions con-
taining the corresponding DNA/RNA target at concentrations 1000, 800, 600, 400, 200, 100,
50, 10, 5, and 1 nM.
RESULTS AND DISCUSSION
Synthesis of Modified Phosphoramidite Reagent
The modified phosphoramidite reagent 3 was synthesized as shown in
Scheme 1. 3-Perylenecarbaldehyde29 was reacted by reductive amination with
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Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
1353
the DMT-protected 2′-amino-α-L-LNA monomer 1 31 to give nucleoside 2.
Subsequent standard phosphitylation afforded the phosphoramidite deriva-
tive 3 (85% overall yield) which was used in automated oligonucleotide
synthesis to prepare series of modified oligonucleotides (Tables I, II). The
sequences of the 12mer oligonucleotides were similar to those used for
studies of pyrene–perylene 2′-amino-LNA FRET pair35. LNA/DNA mixmer
8mer, 9mer and 17mer sequences were adopted from previous studies on
pyrene 2′-amino-LNA 17
.
SCHEME 1
Reagents, conditions and yields: (i) 3-perylenecarbaldehyde, NaBH(OAc)3, 1,2-dichloroethane
(90%); (ii) NC(CH2)2OP(N(i-Pr)2)2, diisopropylammonium tetrazolide, DCM (94%); (iii) DNA
synthesizer (95–99%). R = perylene-3-yl-methyl; fluorescent dye R is also indicated with rays;
DMT = 4,4′-dimethoxytrityl; T = thymin-1-yl
Synthesis of modified oligonucleotides was performed following standard
protocols, except for incorporation of monomer T* and LNA monomers.
The coupling efficiencies of standard amidites based on the absorbance of
the dimethoxytrityl cation released after each coupling varied between 98
and 100%. While incorporating LNA monomers extended coupling time
(10 min) using 1H-tetrazole as activator was applied giving high coupling
yields (98–99%). However using this procedure we obtained only 78–80%
coupling yield for modified monomer T*. We solved this problem by addi-
tional extension of monomer T* coupling time to 30 min and using of pyri-
dine hydrochloride as an activator. This resulted in 95–99% stepwise
coupling yields of monomer T*. All synthesized oligonucleotides were puri-
fied by RP-HPLC and their identity confirmed by MALDI-TOF mass spec-
trometry and their purity by ion-exchange HPLC (see Experimental).
Hybridization and UV-Vis absorption properties of 2′-N-(perylen-3-yl)-
methyl-2′-amino-α-L-LNA monomer T* within 12mer synthetic oligo-
nucleotides were studied in a medium salt phosphate buffer (100 mM
sodium chloride, 10 mM sodium phosphate, 0.1 mM EDTA, pH 7.0). In all
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1354
Astakhova, Kumar, Wengel:
cases, the denaturation curves displayed sigmoidal monophasic transitions
with a shape similar to that observed for unmodified reference duplexes.
Thermal denaturation and UV-visible absorption data of 12mer probes and
their duplexes with complementary DNA are shown in Table I (spectra not
shown). The perylene-modified oligonucleotides displayed higher affinity
towards complementary DNA than complementary RNA. As can be seen
from data presented in Table I, single incorporation of monomer T* results
in remarkable stabilization of duplexes with DNA targets (∆Tm +5.5, +8.0
and +7.0 °C). Moreover, attachment of perylene to 2′-amino-α-L-LNA re-
sults in significantly higher binding affinity than previously introduced
diastereoisomeric 2′-amino-LNA 35. On the other hand, similar stabilization
effect to modification T* was previously reported for pyrene 2′-amino-
α-L-LNA analogue which was proposed to intercalate into the double he-
lix36. This was also confirmed by red-shifted pyrene absorbance upon hy-
bridization with complementary DNA. However we did not observe any sig-
nificant changes in absorbance spectra of ON1–ON3 upon their binding to
DNA (Table I)37. The perylene fluorophore for both single strands and du-
plexes absorbs with a maximum at 454 nm, an additional band at 419 nm
and a shoulder at 396 nm. We speculate that in case of monomer T*
perylene moiety can be placed in the major groove of the double helix
TABLE I
Thermal denaturation temperatures and absorption maxima of the single-labeled oligo-
nucleotides ON1–ON3 and their duplexes with complementary DNA
Tm (∆Tm),°C
Duplex with complementary
λabs
max
SSP/Duplex with
complementary
DNA/RNA
Oligo-
nucleotide
Sequence, 5′→3′
DNA
RNA
ON1
ON2
ON3
ON4a
CTA GT*A TAG TAC
CTA GTA T*AG TAC
CTA GTA TAG T*AC
CTA GTA TAG TAC
38.0 (+5.5)
40.5 (+8.0)
39.5 (+7.0)
32.5
33.0 (+2.0)
34.5 (+3.5)
33.5 (+2.5)
31.0
454/454/454
454/454/454
454/454/454
–
a
Unmodified reference oligonucleotide. Thermal denaturation temperatures Tm (°C) (change
in Tm per modification relative to corresponding reference duplex, ∆Tm/mod (°C)).
Tm values measured as the maximum of the first derivatives of the melting curves (A260 vs
temperature). All Tm and absorbance maxima (λabsmax) values were recorded in medium salt
phosphate buffer. Reported Tm values and absorbance maxima are averages of at least two
measurements. T* = 2′-N-(perylen-3-yl)methyl-2′-amino-α-L-LNA monomer; SSP = single
stranded probe
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Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
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since this type of orientation has been also previously proposed for N-
functionalized 2′-amino-α-L-LNA 36
.
Next, we investigated fluorescent properties of monomer T* in dual-
probe and double-labeled probe formats which are often applied in nucleic
acid diagnostic settings (Fig. 2). In a dual-probe format signal generation
requires simultaneous hybridization of two 5′- and 3′-labeled probes in
neighboring regions on a nucleic acid target, whereas in a double-labeled
FIG. 3
Steady-state fluorescence emission spectra of a ON5 and b ON6+ON7 and the corresponding
duplexes with complementary DNA/RNA. Spectra were obtained in a medium salt phosphate
buffer at 19 °C using an excitation wavelength of 425 nm and 0.1 µM concentration of
oligonucleotides and target strands
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1356
Astakhova, Kumar, Wengel:
probe which is a covalently linked analogue of the dual-probe fluorescence
detection of hybridization is provided by interactions of two consequently
incorporated reporting groups38,39
.
Steady-state fluorescence emission spectra of oligonucleotides ON5–ON7
and their duplexes with DNA/RNA targets were obtained in medium salt
phosphate buffer using 0.1–0.5 µM concentration of the single stranded
probes or the two complementary strands (Fig. 3). Table II lists the resulting
spectral and photophysical properties of single stranded LNA/DNA mixmer
oligonucleotides (SSP) and their duplexes with complementary DNA and
RNA strands.
For the recording of fluorescence spectra an excitation wavelength of
425 nm corresponding to the second perylene absorption maximum was
used. As one can see, the emission peaks of perylene 2′-amino-α-L-LNA
probes lie within the 440–590 nm region, and all the spectra exhibit signifi-
cant vibronic features (Fig. 3). Single stranded probes in both dual-probe
and double-labeled formats display emission bands at fluorescence maxima
λfl ≈ 453 nm, which is approx. 60 nm red shifted relative to the correspond-
ing probes containing pyrene 2′-amino-α-L-LNA 27. Upon duplex formation
the fluorescence maximum of ON5 shifts 9–10 nm to shorter wavelengths
which points on direction of the two fluorophores to the less polar
microenvironment compared to the aqueous buffer25,40,41. Emission max-
ima of the dual-probe system ON6+ON7 on the contrary are only slightly
TABLE II
Spectroscopic and photophysical properties of modified double-labeled and dual-probe
oligonucleotides and their duplexes with complementary DNA and RNA targets
λflmax, nm
ΦF
FB
Oligo-
nucleotide
SSP
DNA
RNA
SSP
DNA
RNA
SSP
DNA
RNA
ON5
454
452
444
451
443
451
0.21
0.91
0.42
0.45
0.35
0.47
12.8
39.8
33.1
16.5
24.3
17.3
ON6+ON7
Modified LNA/DNA mixmer oligonucleotides prepared in this study: ON5: 5′-d(CALC CAA
CT*T* CTLT CCA CLA), ON6: 5′-d(CALC CAA CT*), ON7: 3′-d(ACLA CCT TLCT*), where
AL, TL and CL are adenine-9-yl, thymin-1-yl and 5-methylcytosin-1-yl LNA monomers, re-
spectively. λflmax, ΦF and FB are fluorescence maximum, fluorescence quantum yield and flu-
orescence brightness values, respectively (FB = ΦF × εmax, where εmax is a molar absorption
coefficient measured at the corresponding absorption maximum). SSP = single stranded
probe
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Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
1357
affected by hybridization. Based on these results we propose that a covalent
link between two T* monomers in the double-labeled probe provides a re-
markable degree of rigidity in the double helix which enforces positioning
of the bulky perylene moieties in the groove.
Fluorescence emission quantum yields of oligonucleotides and duplexes
were determined by the relative method using highly diluted solution of
perylene (ΦF = 0.93) as a reference standard42. Interestingly, quantum yields
of modified oligonucleotides and duplexes were not affected by presence of
air in the examined samples, since series of degassed probes gave exactly
the same ΦF values as those in aerated buffer solutions. This is most likely
caused by short fluorescence lifetime of perylene decreasing the possibility
of quenching by solvent, nucleobases or oxygen in the sample solutions.
Superiority of perylene relative to pyrene with respect to quantum yield be-
comes clear if fluorescence rate constants are compared (Kf = 0.25 × 10–7 s–1
for pyrene and 14.62 × 10–7 s–1 for perylene in cyclohexane, respectively42).
Generally, fluorescence quantum yields of ON5–ON7 and their duplexes
are remarkably high (Table II). Usually, polyaromatic hydrocarbons, e.g.
pyrene and its derivatives incorporated into nucleic acids, display low
quantum yields due to interactions with quenchers of fluorescence, e.g. sol-
vent molecules and nucleobases43. However, if the sequence can adopt
FIG. 4
Representative target titration curve for duplex of ON5 and complementary DNA. Spectra were
obtained in a medium salt phosphate buffer at 19 °C using an excitation wavelength of 425
nm and 500 nM concentration of the probe
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1358
Astakhova, Kumar, Wengel:
a structure shielding the fluorophore from the quenchers (i.e. oxygen,
nucleobases), quantum yields of oligonucleotides become very high26. Di-
rection of the fluorophore into the groove upon hybridization can also
reduce fluorescence quenching26,44. The higher quantum yield of single
stranded ON6+ON7 relative to ON5 might result from an interaction be-
tween the two perylene moieties in the latter case which quenches fluores-
cence. Furthermore, single-stranded probe ON5 having two incorporations
of monomer T* exhibit lower quantum yield compared to its duplexes with
DNA and RNA targets (Table II). The bright green fluorescence exhibited by
the duplex ON5:DNA can be detected by the naked eye at concentrations
below 0.1 µM. Interestingly, incorporation of perylene modifications T*
into a dual-probe format did not result in high values of the quantum
yields for the corresponding probe ON5. This stresses that upon attachment
to 2′-amino-α-L-LNA, the fluorescence properties of perylene are as sensitive
to the probe design as those of pyrene44, and that the optimal probe design
needs to be investigated for each particular nucleotide analogue containing
perylene.
Finally, we determined the limit of target detection34, a crucial parameter
of any diagnostic method, for the LNA/DNA probes containing monomer
T*. The LOD value was determined as a lowest concentration of target in a
dilution series detected by fluorescence at the corresponding λfl
such
max
that the fluorescence signal to noise ratio (S/N) (relative to the blank solu-
tion of a medium salt phosphate buffer) was minimum three. As a result of
the high quantum yields of the perylene 2′-amino-α-L-LNA probes, LOD
values for the probes ON5–ON7 are very low (5–10 nM) (Fig. 4). Such limits
of target detection are comparable with those of molecular beacons con-
taining bright xanthene dyes45 and are satisfactory for direct diagnostics of
PCR amplicons.
CONCLUSIONS
Herein, we have reported the synthesis of 2′-N-(perylen-3-yl)methyl-
2′-amino-α-L-LNA monomer T* and its incorporation into synthetic oligo-
nucleotides, Generally, oligonucleotides containing monomer T* exhibited
high-affinity hybridization towards complementary DNA and RNA with the
highest affinity towards DNA. A double-labeled LNA/DNA probe containing
monomers T* showed considerably quenched fluorescence as a single
stranded probe, and an evident increase of fluorescence quantum yield (up
to 2.5-fold) upon hybridization with complementary DNA and RNA targets.
On the contrary, in the alternative dual-probe format, fluorescence was
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1347–1360

Fluorescent Oligonucleotides Containing 2′-Amino-α-L-LNA
1359
remarkably quenched upon binding of complementary DNA and RNA. No-
tably, oligonucleotide probes containing the novel perylene 2′-amino-
α-L-LNA monomer displayed approximately 60 nm red shifted absorption/
emission maxima compared to pyrene, high fluorescence quantum yields
and fluorescence brightnesses. Target titration experiments showed that
targets in concentrations of 5–10 nM target can be effectively detected using
the probes containing monomer T*. The attractive spectral characteristics
reported herein, together with high-affinity binding to complementary
targets, make probes containing 2′-N-(perylen-3-yl)methyl-2′-amino-
α-L-LNA monomer potentially useful for nucleic acid targeting both in vitro
and in vivo.
The authors are thankful to Ms. J. Hansen and Ms. T. Jensen for assistance with oligonucleotide
synthesis and purification. This work was supported by the Danish National Research Foundation and
the Villum Kann Rasmussen Foundation.
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