Branched DNA nanostructures efficiently stabilised and monitored by novel pyrene-perylene 2′-α-l-amino-LNA FRET pairs

Article abstract of DOI:10.1039/c2cc37547h

Novel pyrene-perylene α-l-LNA FRET pairs described herein effectively detect assembly of 2- and 3-way branched DNA nanostructures prepared by postsynthetic microwave-assisted CuAAC click chemistry. The fluorescent signalling of assembly by internally posi

Full text of DOI:10.1039/c2cc37547h

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Branched DNA nanostructures efficiently stabilised and
monitored by novel pyrene–perylene 20-a-L-amino-LNA
FRET pairs†‡
I. Kira Astakhova,*a T. Santhosh Kumar,a Meghan A. Campbell,a Alexey V. Ustinov,b
Vladimir A. Korshunb and Jesper Wengel*a
Cite this: DOI: 10.1039/c2cc37547h
Received 16th October 2012,
Accepted 19th November 2012
DOI: 10.1039/c2cc37547h
www.rsc.org/chemcomm
Novel pyrene–perylene a-L-LNA FRET pairs described herein
Another important task for modern DNA nanotechnology is
effectively detect assembly of 2- and 3-way branched DNA nano- development of synthetic oligonucleotide junctions which could
structures prepared by postsynthetic microwave-assisted CuAAC bring more freedom to the design of advanced nanostructures.3
click chemistry. The fluorescent signalling of assembly by internally Significant improvement of DNA branching strategies has been
positioned FRET pairs is achieved with low to no fluorescence made possible by development of the copper(I)-catalyzed azide–
background signal, remarkably low limit of target detection values alkyne cycloaddition (CuAAC) click chemistry.4 As recently optimized
and stabilization of the resulting nanostructures.
by M. G. Finn et al. the CuAAC reaction seems to be a perfect
bioconjugation method for oligonucleotides, allowing preparation of
DNA nanotechnology is an evolving field of research which various conjugates in high yields and of remarkable purity.5
applies the excellent recognition abilities of nucleic acids to
Among various fluorescent nucleotide analogues, polyaromatic
prepare nanometer- and Ångstrom-scale structures and devices hydrocarbons (PAHs) attached to 20-amino- and the isomeric 20-a-L-
with potential applications in the rapidly developing areas like amino locked nucleic acids (LNA) have proven to be promising units
molecular diagnostics, drug delivery and material science.1 for DNA nanotechnology.6–8 In the present work we prepared a series
Monitoring the process of self-assembly by a simple, sensitive, of branched DNA nanostructures containing two novel internally
inexpensive and rapid sensing method is an important aspect positioned pyrene–perylene 20-a-L-amino-LNA FRET pairs leading
¨
¨
of current DNA nanotechnology. Recently Forster resonance simultaneously to stabilization and efficient fluorescent sensing of
energy transfer (FRET) has been successfully applied in DNA DNA nano-assemblies (Fig. 1). We report the synthesis of oligo-
nanodevices, e.g. for detection of movement by the bright nucleotides containing pyrene and perylene 20-a-L-amino-LNA
Cy3–Cy5 FRET pair.2 However, in order to further utilize FRET nucleotides and 50-alkyne modifications, their further branching
in DNA nanotechnology, the fluorescent dyes have to meet by microwave-assisted CuAAC click reactions through three types of
several requirements, such as high photo- and chemical stability, 2-and 3-way junctions (6–8), and comprehensive spectroscopic
low fluorescence background signal of single strands and high characterization of the resulting fluorescent nanostructures.
stability of fluorescently modified DNA nanostructures. Ideally,
Based on our previous research data,6–9 we selected perylene-
incorporation of fluorescent dyes should furthermore keep the methyl and pyrenecarbonyl 20-a-L-amino-LNA monomers
30- and 50-termini of the blocks free for further assembly and
conjugation strategies.
a Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University
of Southern Denmark, DK-5230 Odense M, Denmark. E-mail: ias@sdu.dk,
jwe@sdu.dk; Fax: +45 6615 8780; Tel: +45 6550 2510
b Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya
16/10, 117997 Moscow, Russia. Fax: +7 499 7246715; Tel: +7 499 7246715
† This article is part of the ‘‘Nucleic acids: new life, new materials’’ web themed
issue.
‡ Electronic supplementary information (ESI) available: Details of synthesis,
assembly and purification of the oligonucleotides, azide 5 and nanostructures;
MALDI-MS, IE HPLC and GE data; selected thermal denaturation curves,
absorption spectra, fluorescence emission and excitation spectra, quantum
yields, FRET measurements and calculations, and molecular modeling. See DOI:
Fig. 1 Modified monomers M1–M4 applied in this study.
10.1039/c2cc37547h
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M1–M3 for the novel interstrand communication FRET systems
(Fig. 1). Additionally, monomers M2–M3 allowed us to evaluate
influence of rather small variation in linker chemistry between
PAH and the nucleotide on the optical properties of the FRET
pairs. In order to obtain fluorescent nucleotide building blocks,
phosphoramidite reagents 1a–c were prepared and used in an
automated solid-phase oligonucleotide synthesis of comple-
mentary oligonucleotides ON1–ON3 following recently
described procedures (Fig. 1, Table 1; ESI,‡ Table S1).6,7 Com-
mercially available phosphoramidite 2 was used in the last step
of the oligonucleotide synthesis and resulted in incorporation
of the 50-terminal alkyne monomer M4, a reactive ‘‘handle’’ for
further click reactions. Additional LNA monomers were incor-
porated next to the 30 termini of ON1–ON3 to additionally
enhance their binding affinity.
Scheme 1 Postsynthetic CuAAC click chemistry generating branched fluorescent
oligonucleotides.
CuAAC click conjugation of ON1–ON3 with azides 3–5 was
performed under microwave conditions for 15 min at 60 1C giving intermediate.11,12 In the next step of our study, we aimed to prove
the desired branched products C1–C5 in high purity and 63–79% the utility of C1–C5 in the assembly of several fluorescent DNA
yields (Scheme 1, Table 1; ESI,‡ Table S2, Fig. S1). Importantly, PAHs nanostructures (Table 2). Starting from 3-way branched strands
were stable under the reaction conditions, and only acetone pre- C4–C5 and corresponding complements C1–C3 possessing pyrene
cipitation and NAP-10 filtration had to be applied for isolation of the donors and perylene acceptor FRET, respectively, we performed
products. In the prepared constructs, M1 was incorporated into assembly, followed by fluorescence spectroscopy, gel electrophoresis
2-way branched conjugates involving rigid diphenyl and perylenebi- and confirming formation of the desired ‘‘nano-star’’ and ‘‘nano-
simide linkers 6 and 7, prepared following described procedures.10,11 acetylene’’ nanostructures by thermal denaturation experiments
All the fluorescent monomers M1–M3 were furthermore inserted into (Table 2; ESI,‡ Fig. S3–S9). The annealing was performed in sodium
3-way branched constructs linked through triphenylmethyl core 8 phosphate and magnesium containing buffers of various salt con-
(ESI,‡ Scheme S1). Incorporation of the fluorescent monomers into centrations (5–710 mM, pH 7.0) applying rapid and slow annealing
C1–C5 was further confirmed by characteristic bands in the visible regimes described in ESI.‡ Having investigated the influence of
region of the corresponding absorbance spectra obtained in a these parameters on formation of the nanostructures, only a slight
medium of salt phosphate buffer (110 mM Na+, Table 2; ESI,‡ difference in the yields of exclusively formed ‘‘nano-star’’ and
Fig. S2). Fluorescence of the conjugates, except C4, was remarkably ‘‘nano-acetylene’’ products was observed (ESI‡). Interestingly,
quenched (Table 1). The observed quenching occurs, most likely, further polymerization of the DNA motifs, which generally can be
due to exposure of the dyes to the polar media within single- monitored as a slow migrating band on a gel,12 was not observed
stranded complexes,6 and due to interactions between the PAHs under any of the applied conditions (ESI,‡ Fig. S6). This suggests
and the branching units 6–8. The observed quenching effect under- high stability of the rather small nanostructures, most likely, due to
lines the high sensitivity of PAHs to the local microenvironment sterical hindrance and high hydrophobicity of the multiply inserted
including linker chemistry between a nucleotide and PAH, as well as monomers M1–M3. This high stability and the presence of free
between the PAH-modified oligonucleotide chains (Table 1).
hybridization valences open up the possibility to use analogue
Complementarity driven DNA assembly allows convenient elements as core motifs for further assembly of even more diverse
preparation of various DNA nanostructures from smaller synthetic nanostructures.11,12
strands and, importantly, starting from
a
single common
Importantly, using M2/M1 and M3/M1 FRET pairs the assembly
of the ‘‘nano-stars’’ and ‘‘nano-acetylenes’’ could be efficiently
monitored using fluorescence at 450 nm in solution and by gel
with remarkably high discrimination values D between the nano-
structure and the corresponding single-stranded building blocks
(D up to 66.0; Table 2). Interestingly, D values were remarkably
improved by branching of the FRET complexes (e.g. C1:C5
compared to ON1:ON3, Table 2) while only slightly affected by
changing the annealing buffer and temperature regimen (ESI,‡
Fig. S9). By comparison of the position and ratios of visible
absorbance and fluorescence maxima of the prepared conjugates,
single strands and previous examples of PAHs attached to LNA,6–9
we conclude that the pyrene fluorophores intercalate into the
p-stack of the DNA helixes, while the perylene moieties locate in
the major groove, which was further confirmed by molecular
modelling studies of monomer (not branched) double helixes
Table 1 Spectral and photophysical properties of the single-stranded branched
conjugatesa
abs
fl
Starting oligonucleotide
l
l
max
(nm)
max
50-30 (ON#)
Azide Product (nm)
Ff
FB
M4CGTGAM1ATATA
3
4
5
5
C1
C2
C3
C4
447, 418 —
447, 418 —
447, 418 450,485 0.07 6.5
347, 329 400
347, 329 —
0.01 0.5
0.01 0.5
ALA (ON1)
ꢀ
M4TTTATAM2AT
CA5-MeCLG (ON2)
M4TTTꢀATAM3ATCA
0.47 45.3
0.01 0.3
5
C5
5-MeCLG (ON3)
ꢀ
a
lambasx, lmfl ax, Ff and FB are maxima of absorbance, fluorescence,
fluorescence quantum yield and FB (fluorescence brightness) = Ff ꢁ emax
.
Ff values are measured by a relative method (ESI) using 1-pyrenebutyric
acid and perylene as fluorescence standard and lex of 330 nm. LNA
monomers are underlined and marked with an L in superscript.
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hybridization systems13). Value J was 3.4 ꢁ 10ꢀ14 cm6 mmolꢀ1
,
Table 2 Properties of fluorescent nanostructures and reference duplexes in a
medium salt phosphate buffera
which is slightly higher than that for the unmodified dyes and
previous examples of pyrene–perylene FRET pairs within DNA
(2.3 ꢁ 10ꢀ14 cm6 mmolꢀ1).13 Efficient FRET of the novel pairs
was additionally confirmed by excitation spectra of the double-
stranded complexes (ESI,‡ Fig. S7). Finally, the Stokes shift for
M2/M1 and M3/M1 was 120 nm which is superior to that
previously reported for cyanines2 and is an additional attractive
parameter for the prepared FRET pairs especially in relation to
appealing in vitro and in vivo imaging techniques, including
single-molecule FRET studies14a and real-time monitoring of
the nanostructures and nanodevices.14b
In conclusion, the novel pyrene–perylene 20-a-L-LNA FRET
pairs are demonstrated to be excellent and stable fluorescent
units in preparations of branched DNA nanostructures. The
background fluorescence of branched single strands was
significantly reduced, while upon formation of nanostructures
emission increased giving remarkably high quantum yields
(Ff up to 0.61) and, therefore, low limit of detection values
(o10 nM in solution). Importantly, this was achieved by internal
modification of the oligonucleotide building blocks providing
additional thermal stability and free termini of the nano-
structures for further conjugation strategies. Accompanied by a
Stokes shift of 120 nm, effective FRET interactions (E up to 1.00)
and very low to no single-strand background signal (D up to
66.0), the novel pyrene–perylene a-L-LNA FRET pairs have high
potential as advanced units for building stable diverse nanos-
tructures allowing simultaneous monitoring of their assembly
and performance.
abs
fl
Tm,
1C
l
max
l
max
D
Ff FB
E
#
Illustration
(nm)
(nm)
ON1:ON2
28
446, 418 450
3.0 0.10 5.8 0.85
C1:C4
C2:C4
23
27
443, 415 450
443, 415 450
6.0 0.52 49.7 1.00
2.9 0.48 45.5 0.93
C3:C4
40
442, 415 450
3.3 0.61 58.3 0.86
5.4 0.23 12.9 1.00
ON1:ON3
27.5 445, 414 450
C1:C5
C2:C5
38
37
447, 419 450 66.0 0.39 37.2 1.00
447, 419 450
9.3 0.37 35.3 1.00
C3:C5
35
443, 417 450
5.7 0.47 45.0 1.00
a
Tm reference (50-CGTGATATATAAA):(30-TTTATATATCACG) = 32 1C.
For details see Table 1 and ESI. D is determined as a ratio of
fluorescence intensities at 450 nm of a double-stranded complex to that
of the corresponding single-stranded mixture; E is FRET efficiency.
Oligonucleotides containing monomers M1, M2 and M3 are presented
as green, blue and purple arrows, respectively. Synthetic junctions 6–8
are indicated as a circle. Properties of the reference complexes ON1:C4
and ON1:C5 are described in Table S3, ESI.
The authors would like to acknowledge financial support
from THE VILLUM FOUNDATION and The Sapere Aude
programme of The Danish Council for Independent Research.
(e.g. ON1:ON3 model in ESI,‡ Fig. S10). The favorable positioning
of the PAHs within DNA, promoted by attachment to 20-a-L-amino-
LNA via short linkers, furthermore resulted in generally increased
thermal stabilities of the prepared nanostructures, especially those
containing 3-way junction 8 and pyrene-labelled monomer M3
(Table 2, e.g. Tm 40 1C for C3:C4 compared to 27.5 1C for
ON1:ON3). Moreover, placing of the PAHs in non-polar media
upon hybridization resulted in high quantum yields and, there-
fore, remarkably low limit of detection values (LOD) down to o10
nM in nanostructure solution and below 0.1 nmol on a gel (ESI,‡
Fig. S6).
Notes and references
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¨
Finally, evaluation of FRET efficiency E, Forster radius R0
values (the distance between fluorophores at which E = 50%)
and spectral overlap integral J between donor emission and
acceptor absorbance gave a further explanation of the observed
fluorescence effects within the created nanostructures (ESI‡). E
values reached 100% upon assembly of the single-stranded
building blocks corresponding to B3 Å distance between the
two PAHs and favourable dipole orientation of the donor and
acceptor FRET (estimated R0 of M2/M1 and M3/M1 was 21.5 Å
and 20.5 Å, respectively, which is B10 Å less than that for the
unmodified pyrene–perylene FRET pair in cyclohexane (R0
31.3 Å) and is in good agreement with previously reported
=
14 (a) J. Tisler, et al., ACS Nano, 2011, 5, 7893; (b) J. M. Thomas, H. Z. Yu
and D. Sen, J. Am. Chem. Soc., 2012, 134, 13738.
c
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