Novel interstrand communication systems within DNA duplexes based on 1-, 2- and 4-(phenylethynyl)pyrenes attached to 2′-amino-LNA: High-affinity hybridization and fluorescence sensing

Article abstract of DOI:10.1039/c0cc03026k

Functionalisation of 2′-amino-LNA oligonucleotides with 1-, 2- and 4-(phenylethynyl)pyrene fluorophores via a carbonyl linker (PEPyc) resulted in efficient interstrand communication systems in nucleic acid duplexes, providing effective tools for stabilization of nanostructures and fluorescence monitoring of DNA self-assembly.

Full text of DOI:10.1039/c0cc03026k

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COMMUNICATION
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Novel interstrand communication systems within DNA duplexes
based on 1-, 2- and 4-(phenylethynyl)pyrenes attached to
20-amino-LNA: high-affinity hybridization and fluorescence sensingw
Irina V. Astakhova,a Dorthe Lindegaard,a Vladimir A. Korshunb and Jesper Wengel*a
Received 4th August 2010, Accepted 10th September 2010
DOI: 10.1039/c0cc03026k
Functionalisation of 20-amino-LNA oligonucleotides with 1-, 2-
and 4-(phenylethynyl)pyrene fluorophores via a carbonyl linker
(PEPyc) resulted in efficient interstrand communication
systems in nucleic acid duplexes, providing effective tools for
stabilization of nanostructures and fluorescence monitoring of
DNA self-assembly.
Nucleic acids, best known as the carriers of genetic
information, are demonstrated to be highly useful materials
for designing nanometre-scale structures (Angstrom-scale
chemical engineering).1 Oligonucleotides (ONs) have unique
and predictable recognition capabilities due to the specificity
of Watson–Crick base-pairing, making DNA a perfect tool for
growing well-defined nanostructures. Potential applications of
Scheme 1 Synthesis of modified monomers M1–M3 (for details see
the latter include nanoelectronics, biosensors and various
ESIw).
molecular machines. For the preparation of such DNA
nanostructures it should be possible to form stable constructs
following the process of self-assembly by a sensing method of
choice. Stimulated by high-affinity hybridization of LNA
(locked nucleic acid),2 20-amino-LNA3 and its 20-N-
derivatives,4 Hrdlicka et al. introduced 20-N-(pyren-1-yl)-
20-amino-LNAs containing 1-, 2- and 4-PEPy fluorophores
linked via a carbonyl linker (PEPyc-LNA) as improved
fluorescent labels for double-stranded DNA interstrand
communication systems (Scheme 1). Herein, we report the
synthesis, thermal denaturation experiments and fluorescence
properties of ONs containing 1-, 2- and 4-PEPyc-20-amino-
LNAs.
methyl-20-amino-LNA as
a key element in interstrand
communication systems in dsDNA.5 Being incorporated into
short ONs, this monomer provides two effects: stabilization of
duplexes and fluorescence signaling of hybridization. We have
attached various (phenylethynyl)pyrenes (PEPys)6 to 20-amino-
LNA7a as improved fluorescent monomers for nucleic acid
labeling. Among other interesting properties, these conjugates
displayed higher fluorescent quantum yields and long-wave
shifted absorption and emission maxima (by 25–100 nm)
compared to the parent pyrene, which makes them perspective
dyes for nucleic acids labeling and diagnostics.
In order to prepare PEPyc functionalized oligonucleotide
building blocks, phosphoramidite reagents 4a–c were prepared
(Scheme 1). Synthesis of the compounds is described in ESI.w
Attachment of fluorescent dyes to the 20-position of
20-amino-LNA nucleoside was performed by acylation of 18
with p-iodobenzoic acid in the presence of HATU (N,N,N0,N0-
tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluoro-
phosphate)9 followed by Sonogashira coupling10 with 1-, 2-
or 4-ethynylpyrenes11 (Scheme 1). The yields of Sonogashira
couplings correlate with the sterical accessibility of the ethynyl
group in the pyrene derivative: the less hindered 2-ethynyl-
pyrene gave the highest coupling yield (89%), while the most
hindered 4-ethynylpyrene gave only 71% of product 3c.
The nucleoside derivatives 3a–c were then phosphitylated
with bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine in
DCM in the presence of diisopropylammonium tetrazolide
to give phosphoramidites 4a–c, which were used in automated
oligonucleotide synthesis to prepare series of modified ONs.
The sequences of mixed base 9-mer ONs were similar to those
previously designed for studies of 20-N-(pyren-1-yl)methyl-20-
amino-LNA.5
Inspired by this work, and by recent research on 20-N-
(pyren-1-yl)methyl-20-amino-LNA5 and other fluorescent
derivatives of 20-amino-LNA,7 we introduce herein novel
a Nucleic Acid Center, Department of Physics and Chemistry,
University of Southern Denmarkz, DK-5230 Odense M, Denmark.
E-mail: jwe@ifk.sdu.dk; Fax: +45 6615 8780; Tel: +45 6550 2510
b Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
E-mail: korshun@ibch.ru; Fax: +7 495 429 8710;
Tel: +7 495 429 8710
w Electronic supplementary information (ESI) available: Details of
synthesis and purification of modified phosphoramidites and ONs;
MALDI-MS data of modified ONs (Table S1); selected thermal
denaturation curves, absorption spectra, fluorescence emission spectra,
and quantum yields measurements. See DOI: 10.1039/c0cc03026k
z A research center funded by Danish National Research Foundation
for studies of nucleic acid chemical biology.
The effect of modifications M1–M3 on duplex stability was
evaluated by UV thermal denaturation experiments using
medium salt buffer ([Na+] = 110 mM)w and compared to
c
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Table
1
ONs used in this study and representative thermal
Table 2 Representative spectroscopic and photophysical properties
of M1–M3 modified ONs and duplexes with complementary DNA
(ON1) and RNA (ON2)a
denaturation data for ON5–ON13 towards unmodified DNA and
RNA complements (ON1–ON4)a
#
Sequence, 50 - 30
#
Sequence, 50 - 30
lambasx, bands
I–II/nm
#
lflmax/nm
Ff
emax
FB
ON1
ON2
ON3
ON4
ON5
ON6
ON7
GTG ATA TGC
r(GUG AUA UGC)
GCA TAT CAC
r(GCA UAU CAC)
GTG AM1A TGC
GCA M1AT CAC
GCA TAM1 CAC
ON8
ON9
ON10
ON11
ON12
ON13
—
GTG AM2A TGC
GCA M2AT CAC
GCA TAM2 CAC
GTG AM3A TGC
GCA M3AT CAC
GCA TAM3 CAC
—
ON5
372, 395
370, 391
372, 392
305, 325
303, 320
304, 322
351, 364
347, 360
350, 366
455
457
459
402, 430
402, 431
405, 433
422
424
426
0.33
0.37
0.42
0.55
0.72
0.81
0.11
0.16
0.22
38 900
36 000
36 800
51 900
46 000
45 500
32 700
30 000
39 300
12.8
13.3
15.5
28.5
33.8
36.9
3.6
ON5:ON1
ON5:ON2
ON8
ON8:ON1
ON8:ON2
ON11
#
Schematic structure
Tm, 1C
DTm,1C
ON11:ON1
ON11:ON2
4.8
8.7
ON6:ON1
ON6:ON2
37.5
33.5
+7.0
+4.0
lambasx, lflmax, Ff and emax are maxima of absorbance, fluorescence,
fluorescence quantum yield and molar absorption coefficient,
respectively; FB (fluorescence brightness) = Ff ꢁ emax. Ff values are
measured by a relative method (ESIw) using 5-(pyrene-1-ylethynyl)-20-
deoxyuridine and 9,10-diphenylantracene as fluorescence standards.
Ff values are found to be independent on the presence of oxygen in
solutions of samples and all Ff values are therefore presented for
non-degassed samples.
a
ON7:ON1
ON7:ON2
29.5
29.0
ꢀ1.0
ꢀ0.5
ON9:ON1
ON9:ON2
37.0
33.0
+6.5
+3.5
ON10:ON1
ON10:ON2
38.0
36.5
+7.5
+7.0
ON12:ON1
ON12:ON2
40.5
39.0
+10.0
+9.5
unmodified DNA and RNA (Table 2). Hybridization results
in increased fluorescense quantum yields of the dyes and in
slight shifts of absorption and emission maxima. Compared to
analogous constructs containing single incorporations of
20-pyrenemethyl-5 and 20-pyrenecarbonyl-LNA,7c we observe
remarkable shifts of absorption and emission maxima, and
dramatical increases of fluorescence quantum yields and,
hence, of fluorescence brightness values especially for mono-
mer M2. These properties are of significant importance for
fluorescent labels. Thus, controlled shifts of absorption/
emission spectra allow application of monomers M1–M3 in
multiplex formats and in living cells, while high FF values
lower the detection limit.
ON13:ON1
ON13:ON2
37.0
36.0
+6.5
+6.5
a
Melting temperatures were measured in a medium salt buffer using
1.0 mM concentrations of ONs.w The droplets represent PEPyc-LNA
monomers M1 (blue), M2 (red) and M3 (green). DTms are calculated
relative to Tm values of unmodified DNA:DNA (Tm = 30.5 1C) and
DNA:RNA (Tm = 29.5 1C) duplexes.
the stability of the corresponding unmodified reference duplexes
(Table 1). Incorporation of a single PEPyc-20-amino-LNA
modification M1–M3 into a mixed 9-mer DNA sequence
results in high thermal stabilities against both complementary
DNA and RNA (Table 1, DTm values = +3.5 to +9.5 1C)
except for 1-PEPyc containing ON7 (DTm = ꢀ0.5 to ꢀ1 1C).
The resulting Tm values are much higher than those observed
previously for 20-N-(pyren-1-yl)methyl-20-amino-LNA in
analogous constructs, for which molecular modelling showed
positioning of the pyrene units along the minor groove of the
duplexes.5 Thermal stabilities of the duplexes containing
monomers M1–M3 are also much higher, than for previously
reported mono-PEPyc-labeled 20-amino-LNA nucleotides
with alternative arrangement of phenyl- and pyrene-groups.7a
The increased stabilities of the duplexes observed herein
(Table 1) are therefore most likely caused by favorable inter-
actions between the PEPyc units and functionalities in the
minor groove. In the case of monomer M1 the melting
temperatures varied significantly upon insertion of a modification
in position 6 or 4. However, this effect was not observed for the
corresponding ONs containing M2 and M3. The most stable
duplexes were formed by the 4-PEPyc modified ON12 showing a
DTm value of +9.5 1C despite the bulkiness of the appended
fluorophore. We propose that the geometry of the 4-PEPyc
entity linked to a 20-amino-LNA monomer is most favorably
accommodated within the minor groove of the duplexes.
We furthermore studied +1 and ꢀ1 upstream zippery
constructs containing modifications M1–M3 in both
Table 3 Thermal denaturation studies, and spectroscopic and photo-
physical properties of modified double stranded DNA containing
modifications M1–M3 in both complementary strandsa
Schematic
structure
Tm/
Zipper3y DTm 1C
lflmax
/
nm
#
Ff
ON6:ON5
ON7:ON5
ON9:ON8
ON10:ON8
+1
ꢀ1
+1
ꢀ1
64.0/+16.8 455
63.0/+16.3 512
60.0/+14.8 430
62.0/+15.8 435
0.52
0.61
0.87
0.92
ON12:ON11
ON13:ON11
a
+1
61.0/+15.3 430
0.35
0.40
ꢀ1
65.0/+17.3 490
For conditions see Tables 1–2. Ds are given per modification and
calculated relative to the Tm value of unmodified double-stranded
DNA (Tm = 30.5 1C).
Next, we examined spectral and photophysical properties of
the modified ONs and their duplexes with complementary
c
This journal is The Royal Society of Chemistry 2010
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complementary strands (Table 3). Previously it was shown
that insertion of two pyrenylmethyl-20-amino-LNA monomers
in analogous constructs results in stable duplexes which dis-
play intensive fluorescence signals resulting from pyrene ex-
cimer formation.5 In case of M1–M3, complementary pairs of
modified ONs formed highly stable duplexes in both types of
zipper constructs. Such high DTm values imply effective
stabilizing interactions between fluorophores, which is also
confirmed by the appearance of an excimer signal in the
fluorescence spectra for ꢀ1 upstream constructs containig
1- and 4-PEPyc-20-amino-LNA, or strongly stabilizing inter-
actions between fluorophores and the minor groove of the
duplexes. An excimer band was not detected either for their
+1 zippers or for the 2-PEPyc isomer. Similarly, an excimer
signal was previously not observed neither for a +1 upstream
zipper containing pyrenylmethyl-20-amino-LNA monomers5
nor for analogous constructs having different PEPyc-20-
amino-LNAs.7a A +1 zipper constitution thus seems to be
unfavorable for positioning the dyes within a short distance
as needed for excimer formation. As 1-, 2- and 4-PEPy
fluorophores are known to display very short fluorescence
lifetimes on DNA (B 4–15 ns),12 we propose that excimer
formation depends on a preorganized structure of the duplex
rather than reorganization of a structure not predisposed for
excimer formation. We determined a significant increase in
fluorescence quantum yields for all +1 zippers compared to
their singly labeled analogues (Tables 2 and 3). We explain
these data by generally short fluorescence lifetimes of PEPy
dyes attached to DNA,6b,12 and also by their precise positioning
within duplexes typically resulting from their attachment to
20-amino-LNA via a short linker.7 The latter may result in
decreased interaction of fluorophores with such quenchers of
fluorescence as medium, DNA nucleobases and oxygen, and
hence, may increase fluorescence quantum yields of the PEPy
dyes.7c
affinity of hybridization. Upon attachment of phenylethynyl
substituents, the absorption/emission maxima are drastically
shifted, while fluorescence quantum yields are remarkably
enhanced. Besides useful for diagnostic applications, we
propose that excimer formation within 1- and 4-PEPyc
labelled duplexes and the remarkable increase in fluorescence
quantum yields of the 2-PEPyc isomer, makes the three PEPyc
20-amino-LNA monomers useful as advanced signalling units
to monitor self-assembly of nucleic acid nanostructures.
We thank The Sixth Framework Programme Marie Curie
Host Fellowships for Early Stage Research Training under
contract number MEST-CT-2004-504018, The Villum Kann
Rasmussen Foundation and The Danish National Research
Foundation for financial support. V.A.K. was supported by
the Molecular and Cellular Biology Program of the presidium
of Russian Academy of Sciences and Dynasty Foundation.
Notes and references
y The ‘‘zipper’’ nomenclature for defining arrangements of modified
duplexes containing monomers in both complementary strands will be
used in this article. An ‘‘n zipper’’ describes the arrangement of
two modified nucleotides of interest, positioned in opposite strands
of a DNA duplex. The number n indicates the distance in base
pairs between the two nucleotides. If n is positive, the two modified
monomers are positioned relatively toward the 50-end of the
two strands, and if n is negative, the two modified monomers
are positioned relatively toward the 30-end of the two strands.
1 Some recent examples: (a) T. Liedl, B. Hogberg, J. Tytell,
D. E. Ingber and W. M. Shih, Nat. Nanotechnol., 2010, 5,
520; (b) J. B. Lee, M. J. Campolongo, J. S. Kahn, Y. H.
Roh, M. R. Hartman and D. Luo, Nanoscale, 2010, 2,
188; (c) E. S. Andersen and J. Kjems, et al., Nature, 2009,
459, 73.
2 M. Petersen and J. Wengel, Trends Biotechnol., 2003, 21, 74.
3 S. K. Singh, R. Kumar and J. Wengel, J. Org. Chem., 1998, 63,
10035.
4 M. D. Sørensen, M. Pedersen and J. Wengel, Chem. Commun.,
2003, 2130.
5 P. J. Hrdlicka, B. R. Babu, M. D. Sørensen and J. Wengel, Chem.
Commun., 2004, 1478.
In addition, we monitored hybridization of 1-, 2- and
4-PEPyc containing complementary ONs by changes in
fluorescence colour of solutions (Fig. 1). As a result of high
fluorescense brightness values, the formation of duplexes can
be monitored by fluorescence change at sample concentrations
down to approx. 1 nM using a fluorescense spectrometer and
down to 50 nM by the nacked eye.
6 (a) H. Maeda, T. Maeda, K. Mizuno, K. Fujimoto, H. Shimizu
and M. Inouye, Chem.–Eur. J., 2006, 12, 824; (b) I. V. Astakhova,
A. D. Malakhov, I. A. Stepanova, A. V. Ustinov, S. L. Bondarev,
A. S. Paramonov and V. A. Korshun, Bioconjugate Chem., 2007,
18, 972.
7 (a) I. Astakhova, V. Korshun and J. Wengel, Chem.–Eur. J., 2008,
14, 11010; (b) P. Gupta, N. Langkjær and J. Wengel, Bioconjugate
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M. D. Sørensen, N. Harrit and J. Wengel, J. Am. Chem. Soc.,
2005, 127, 13293.
In conclusion, ONs containing novel PEPyc-LNA monomers
M1–M3 are demonstrated to be promising fluorescent building
blocks due to significant improvement of pyrene’s spectral
and photophysical characteristics accompanied by increased
8 (a) C. Rosenbohm, S. M. Christensen, M. D. Sørensen,
D. S. Pedersen, L. Larsen, J. Wengel and T. Koch, Org. Biomol.
Chem., 2003, 1, 655; (b) S. M. Christensen, C. Rosenbohm,
M. D. Sørensen, L. Larsen, J. Wengel and T. Koch, Nucleosides,
Nucleotides Nucleic Acids, 2003, 22, 1131.
9 (a) L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397;
(b) L. A. Carpino and M. Bienert, et al., Angew. Chem., Int. Ed.,
2002, 41, 441.
10 K. Sonogashira, in Comprehensive Organic Synthesis, ed.
B. M. Trost and I. Fleming, Pergamon Press, Oxford, New York,
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11 V. Filichev, I. Astakhova, A. Malakhov, V. Korshun and
E. Pedersen, Chem.–Eur. J., 2008, 9, 9968.
Fig. 1 Photographs showing fluorescence in medium salt buffer of the
two complementary ONs before (vials 1, 3, 5) and after annealing
(vials 2, 4, 6): ON7:ON5 (vials 1–2); ON10:ON8 (vials 3–4),
ON13:ON11 (vials 5–6) (0.25 mM solutions). Photographs were taken
with a digital camera using a laboratory UV-lamp (lex = 365 nm).
12 I. V. Astakhova and V. A. Korshun, Russ. J. Bioorg. Chem., 2008,
34, 510.
c
8364 Chem. Commun., 2010, 46, 8362–8364
This journal is The Royal Society of Chemistry 2010