Programmed assembly of organic radicals on DNA

Article abstract of DOI:10.1039/b913061f

Nitronyl nitroxide radical introduced to naphthyridine carbamate dimer is noncovalently bound to a CGG/CGG triad as an addressable position in DNA duplexes, leading to the programmed assembly of the radical molecules into an 11-mer duplex and a tandem rep

Full text of DOI:10.1039/b913061f

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Programmed assembly of organic radicals on DNAw
Kensuke Maekawa,a Shigeaki Nakazawa,b Hiroshi Atsumi,a Daisuke Shiomi,b
Kazunobu Sato,b Masahiro Kitagawa,c Takeji Takuib and Kazuhiko Nakatani*a
Received (in Cambridge, UK) 3rd July 2009, Accepted 17th December 2009
First published as an Advance Article on the web 18th January 2010
DOI: 10.1039/b913061f
Nitronyl nitroxide radical introduced to naphthyridine carbamate
dimer is noncovalently bound to a CGG/CGG triad as an
addressable position in DNA duplexes, leading to the programmed
assembly of the radical molecules into an 11-mer duplex and a
tandem repetitive array of double stranded DNA.
for controlled functionalities. Here we report a DNA building
block holding two stable organic radicals and its assemblage
into a one-dimensional DNA array. The DNA unit described
here has a sticky DNA overhang, which enables us to organize
the unit into multi-dimensional DNA structures with the
accessible complementary sequence.
We have synthesized a mismatch binding ligand holding
nitronyl nitroxide (NCDNN). The parent mismatch binding
ligand NCD (naphthyridine carbamate dimer) specifically
binds to a G–G mismatch in a 50-CGG-30/50-CGG-30 triad
in the ratio of NCD : triad = 2 : 1 and improved the thermo-
dynamic stability of the duplex.6 We anticipate that the
binding of NCDNN to the CGG/CGG triad enables
programmed assembling of the radical molecules at the
designed position in a DNA structure (Fig. 1). NCDNN was
synthesized by the reductive amination of p-formylphenyl
nitronyl nitroxide with NCD. The thermal denaturation
experiments of 11-mer DNA duplexes 50-(CTAACXGAATG)-
30/50-(CATTCYGTTAG)-30, in which the symbols X and
Y designate A, G, C, or T, were conducted to evaluate the
thermodynamic stability of each duplex in the absence and
presence of NCDNN. The presence of NCDNN led to a
remarkable change of Tm for the duplex containing the G–G
mismatch compared with the others, giving the difference of
Tm of 13.5 1C (DTm) (Table S1w). These results suggest the
DNA molecules have been rediscovered as building blocks for
constructing nanoscale objects by utilizing the sequence-
specific hybridization of complementary strands.1 DNA-based
architectures can provide a well-defined programmable template
with addressable and accessible base sequences,2 exploring the
possibility to assemble auxiliary molecules and functional
groups on multi-dimensional DNA nanostructures.
Stable organic radicals have so far been exploited as spin
probes in spin labeling chemistry to obtain the structural and
dynamic insights into biologically important biomolecules.3
Spin labeling for DNA by the covalent modification of
nucleobases or the phosphate–sugar backbone has also been
actively investigated.4 To utilize the programmable DNA
sequences, we have studied the non-covalent assembly of
stable organic radicals on DNA arrays. The non-covalent
assembly strategy has an advantage over the covalent introductions
of stable organic radicals, as exploited in conventional spin
labeling chemistry. DNA provides a variety of scaffolds for
ligand bindings, e.g. a minor groove binding, an intercalation,
and a major groove binding with a sequence specific manner.
The combination of the modes of the ligand bindings
and stable radical families such as nitroxides, and imino-
or nitronyl nitroxides allows us to obtain a diversity of
DNA-stable radical assembly with well-identified distances
and the different spatial orientations of magnetic tensors,
which can afford scalable electron spin-qubit systems for
quantum computers/quantum information processing systems
based on molecular spins.5 The non-covalent strategy will
provide rapid access to the assemblages of organic radicals
a Department of Regulatory Bioorganic Chemistry,
The Institute of Scientific and Industrial Research (ISIR),
Osaka University, Ibaraki 567-0047, Japan.
E-mail: nakatani@sanken.osaka-u.ac.jp; Fax: +81 6 6879 8459;
Tel: +81 6 6879 8455
b Departments of Chemistry and Materials Science, Graduate School
of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585,
Japan
c Department of System Innovation, Graduate School of Engineering
Science, Osaka University, Toyonaka 560-8531, Japan
w Electronic supplementary information (ESI) available: Synthetic
details of NCDNN, Job’s plot in CD spectra, gel electrophoresis,
simulated parameters and displacement assay for ESR spectra, four-
pulse ELDOR (DEER) experimental data and the spin–spin distance
distributions from molecular dynamics simulations, and AFM image.
See DOI: 10.1039/b913061f
Fig. 1 (a) The structure of NCDNN. (b) The schematic representation
of bindings of NCDNN to the CGG/CGG triad and the DNA unit
embedded with the radicals. (c) The programmed assembly of the
organic radicals on a tandem repetitive array of the double stranded
DNA organized by sticky overhangs.
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selective binding of NCDNN to the mismatched duplex
containing the CGG/CGG triad.
Titration experiments in circular dichroism (CD) spectra
with the 11-mer DNA containing the CGG/CGG triad
showed the increase of the induced CD bands with a negative
and positive Cotton signal as the concentration of NCDNN
increased (Fig. 2). The NMR structure of the complex com-
posed of NCD and a duplex containing the CGG/CGG triad
revealed that the two naphthyridine rings of two NCDs were
stacked along the duplex with each other.7 An exciton band
originating from the stacking structure of the naphthyridine
rings was in fact observed in the CD spectra. The exciton band
has also been found around 328 nm. Job’s plot analysis
clearly showed the binding stoichiometry of NCDNN to the
CGG/CGG triad to be 2 : 1 (Fig. S1w). The concentration
dependence of molar ellipticity at 347 nm was fitted on the
basis of an identical and independent two-site binding model
to estimate a binding constant Ka.8 The fit to the observed
values gave Ka to be 1.6 ꢁ 105 Mꢂ1 with the 2 : 1 stoichiometry
of NCDNN to DNA. These results indicate that (1) the
binding mode of NCDNN to the CGG/CGG triad is similar
to that of NCD and (2) two nitronyl nitroxide radicals bind to
the triad in 105 affinity.
Fig. 3 ESR spectra of NCDNN (200 mM) in a buffer containing
100 mM NaCl, 10 mM sodium cacodylate (pH 7.0) and 10% (v/v)
methanol in the (a) absence and (b) presence of the 11-mer DNA
duplex (100 mM) at 297 K with microwave frequency nMW
=
9.44601(7) GHz for (a) and 9.44482 (4) GHz for (b). The dashed lines
represent simulated spectra. (c) A spectrum of NCDNN (200 mM) in a
sodium cacodylate buffer (10 mM, pH 7.0) containing the 25-mer
DNA strands (100 mM), 100 mM NaCl and 10% (v/v) methanol at
276 K with nMW = 9.44849(2) GHz.
Fig. 3a and b show solution ESR spectra of NCDNN in the
absence and presence of the 11-mer duplex containing
the CGG/CGG triad, respectively. The spectrum of only
NCDNN exhibited a typical hyperfine splitting pattern with
equally separated five peaks attributed to the two nitrogen
nuclei on the radical moiety. The spectrum of the radical
solution containing the duplex showed a remarkable change
featuring in a line broadening dependent on the mI component
of the nuclear spin I.9 The broadening reflects a slower
tumbling of the radical moieties compared with that of only
the NCDNN. No electron spin forbidden transition with
Dms = ꢃ2 was observed for the duplex-containing sample in
the frozen glass at 143 K. Both the observed spectra were
simulated to evaluate a rotational correlation time tc assuming
an isotropic rotational diffusion in a fast regime and only an
S = 1/2 spin state. Due to the equilibrium of NCDNN
binding to the CGG/CGG triad, the fraction of the bound
form (a) was taken into account for the simulation. The
simulations reproduced the experimental data as the para-
meters tc = 6.7 ꢁ 10ꢂ11 s for only the radical (Fig. 3a) and
9.1 ꢁ 10ꢂ10 s for the DNA-bound radicals with a = 0.88
(Fig. 3b) where the correlation time of DNA-free components
is fixed as the former value. These results indicate that the
tumbling of the radical moieties embedded in the duplex is
slower than that of the unbound NCDNN. Under the con-
centration conditions, the fraction of 0.88 is in good agreement
with that of 0.83 calculated from the association constant
obtained from the CD titration. The line shape in the spectrum
of the radical-DNA system approximated to that of only the
radical by the displacement of NCDNN with NCD, indicating
a competitive binding between NCD and NCDNN (Fig. S2w).
Encouraged by these findings, a radical-DNA complex
has been expanded into a one-dimensional DNA system
consisting of two 25-mer DNA strands 50-(GCACTCGGA-
CAGATTCTTGAGCTCT)-30/50-(TCTGTCGGAGTGCAG-
AGCTCAAGAA)-30, leading to the tandem repetitive array
of double stranded DNA by the hybridization on sticky over-
hangs (Fig. 1c). Smear bands observed for the duplex on
native PAGE analyses and the mobility shift with NCDNN
showed the formation of one-dimensional DNA arrays
holding the radical molecules (Fig. S3w). The intensity for
the smear band of only the DNA array has the maximum at
200 bp in length with a dispersion (Fig. S3(b)w). As shown in
Fig. 3c, the ESR spectrum of the NCDNN solution containing
the 25-mer DNA strands exhibited an anisotropic feature
more prominently than that of the radicals embedded in
the 11-mer duplex, which is due to the size effect of
the DNA array. These results have demonstrated the
Fig. 2 (a) CD spectra for the titration of the DNA duplex (4.5 mM) in
sodium cacodylate buffer (10 mM, pH 7.0) containing 100 mM NaCl
and 10% (v/v) methanol with NCDNN. Key: [NCDNN] = 0, 4.5, 9,
18, 36, 45, 63, 72 mM. The arrows designate the change of a molar
ellipticity with increased concentration of NCDNN. (b) Concentration
dependence of the molar ellipticity at 347 nm. The closed circles and
the solid line denote the observed values and the theoretical curve
based on an identical and independent two-sites binding model.
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self-assembly of the stable organic radicals on the one-
dimensional DNA array.
Notes and references
1 N. C. Seeman, Nature, 2003, 421, 427; P. W. Rothemund, Nature,
2006, 440, 297.
2 J. D. Cohen, J. P. Sadowski and P. D. Dervan, Angew. Chem., Int.
Ed., 2007, 46, 7956; J. D. Cohen, J. P. Sadowski and P. D. Dervan,
J. Am. Chem. Soc., 2008, 130, 402.
3 Spin Labeling, ed. L. J. Berliner, Academic Press, New York, 1976;
Spin Labeling II, ed. L. J. Berliner, Academic Press, New York,
1979; G. I. Likhtenstein, Spin Labeling Methods in Molecular
Biology, Wiley Interscience, New York, 1976.
4 For recent examples of oligonucleotides labeled with stable
nitroxides, see: A. Okamoto, T. Inasaki and I. Saito, Tetrahedron
Lett., 2005, 46, 791; N. Barhate, P. Cekan, A. P. Massey and S. Th.
Sigurdsson, Angew. Chem., Int. Ed., 2007, 46, 2655; G. Sicoli,
G. Mathis, O. Delalande, Y. Boulard, D. Gasparutto and
S. Gambarelli, Angew. Chem., Int. Ed., 2008, 47, 735;
O. Schiemann, P. Cekan, D. Margraf, T. F. Prisner and S. Th.
Sigurdsson, Angew. Chem., Int. Ed., 2009, 48, 3292.
5 K. Sato, S. Nakazawa, R. Rahimi, T. Ise, S. Nishida, T. Yoshino,
N. Mori, K. Toyota, D. Shiomi, Y. Yakiyama, Y. Morita,
M. Kitagawa, K. Nakasuji, M. Nakahara, H. Hara, P. Carl,
P. Hofer and T. Takui, J. Mater. Chem., 2009, 19, 3739, and
reference therein: For electron spin-qubits, the different spatial
orientation of non-equivalent g-tensors is relevant. This is termed
g-tensor engineering.
6 T. Peng and K. Nakatani, Angew. Chem., Int. Ed., 2005, 44, 7280;
T. Peng, C. Dohno and K. Nakatani, Angew. Chem., Int. Ed., 2006,
45, 5623; T. Peng, C. Dohno and K. Nakatani, ChemBioChem,
2007, 8, 483.
7 M. Nomura, S. Hagihara, Y. Goto, K. Nakatani and C. Kojima,
Nucleic Acids Symp. Ser., 2005, 49, 213.
8 K. A. Connors, Binding Constants—The Measurements of
Molecular Complex Stability, John Wiley & Sons, Toronto, 1987.
9 D. Kivelson, J. Chem. Phys., 1960, 33, 1094.
To estimate the intermolecular spin–spin distance between
the radicals embedded within the NCDNN/DNA complex
(11-mer duplex) in a straightforward manner is of particular
interest. Q-band four-pulse ELDOR (DEER) spectroscopy10
applied to the complex gave two dominant distances at 1.8 and
2.0 nm (Fig. S4w). The latter was more dominating. Also, we
have invoked molecular dynamics simulations for a model
complex in which the radical sites are replaced by carbonyl
groups,10 acquiring the distributions of the distances
(Fig. S5–S6w). Two dominating distances of 1.7 nm and
1.9 nm appeared and the latter is more prominent. The
observed distances are in harmony with these calculated
values, indicating that the radicals of the naphthyridine
carbamate dimer are bound to the CGG/CGG triad as the
addressable positions in the DNA duplexes.
The newly synthesized NCDNN is bound to the CGG/CGG
triad in 105 affinity and delivered the two radicals on the DNA
unit. The self-assembly of the DNA unit with sticky overhangs
produced one-dimensional DNA arrays with multiple spins
on. The DNA-spin unit would be useful for the construction of
two- and three-dimensional DNA structures holding mole-
cular spins at the programmed positions. The addressability of
radical spins is of particular importance for spin–spin mutual
microscopic arrangements in a controlled manner such as
construction architecture for molecular spin-based quantum
computers with a DNA backbone.5,10
10 The carbonyl compounds in which the NO radical sites are
substituted by CO groups can well corporate, at any desired
concentrations, the corresponding NO radicals in the crystal
lattices of the carbonyl molecules, indicating crystal-engineering
similarity between the two compounds: S. Nakazawa, T. Ise,
T. Nishida, T. Yoshino, N. Mori, K. Toyota, D. Shiomi,
Y. Morita, M. Kitagawa, H. Hara, P. Carl, P. Hoefer and
T. Takui, unpublished work.
This work has been supported by Grants-in-Aid for Scientific
Research (S) (18105006) for K.N. and (B) (19350072) for D.S.
from MEXT, and also by Core Research for Evolutional Science
and Technology (CREST-JST). One of the authors (K.M.)
acknowledges Research Fellowships (20–2047) of the Japan
Society for the Promotion of Science for Young Scientists.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 1247–1249 | 1249