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© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2808
NATURE CHEMISTRY | www.nature.com/naturechemistry 1
1
A metallo-DNA nanowire with uninterrupted
one-dimensional silver array
Jiro Kondo1,2,*, Yoshinari Tada2, Takenori Dairaku3, Yoshikazu Hattori4, Hisao Saneyoshi5, Akira Ono5, Yoshiyuki Tanaka4
1 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan. 2 Graduate School of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan. 3 School of Pharmaceutical Sciences, Ohu University, 31-1 Misumido, Tomita-machi, Koriyama, Fukushima 963-8611, Japan. 4 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, 770-8514 Tokushima, Japan.
5 Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan. * Corresponding Author: Dr. Jiro Kondo Tel: +81-3-3238-3290, Fax: +81-3-3238-3361 E-mail address: j.kondo@sophia.ac.jp
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Methods Crystallization. The DNA dodecamer with a sequence d(GGACT[
BrC]GACTCC) (
BrC =
5-bromo-2’-deoxycytidine) designed to form a self-complementary duplex containing two CC
mismatches was chemically synthesized (Gene Design). The Br
C residue was introduced to resolve
the phase problem by the anomalous dispersion method. This DNA was purified by denatured 20%
polyacrylamide gel electrophoresis at the condition containing 3.2 M urea and then desalted by
reversed phase chromatography. Crystallizations were performed by the hanging-drop vapour
diffusion method at 293 K in the presence of silver ions. Prior to crystallization, 4 mM DNA solution
was mixed with the same volume of 8 mM silver nitrate. Crystallization droplets were prepared by
mixing 1 l of sample solutions and 1 l of crystallization solutions containing 50 mM
3-morpholinopropanesulfonic acid (MOPS) (pH 7.0), 10 mM spermine, 10% (v/v)
2-methyl-2,4-pentanediol and 10-500 mM cation nitrates. The droplets were equilibrated against
reservoir solutions containing 40% (v/v) 2-methyl-2,4-pentanediol. Single crystals were grown in
conditions containing potassium nitrate. The optimized crystallization conditions are summarized in
Supplementary Table 1. Fresh crystals were mounted in nylon cryoloops (Hampton Research) with
the crystallization solution that contained 40% (v/v) 2-methyl-2,4-pentanediol as a cryoprotectant
and stored in liquid nitrogen prior to the X-ray experiments.
Data collection, structure determination and refinement. X-ray datasets were collected at 100K
with synchrotron radiation at the BL-5A and BL-1A beamlines in the Photon Factory in Tsukuba,
Japan. An X-ray dataset collected for the single wavelength anomalous dispersion (SAD) phasing
was processed by the program CrystalClear (Rigaku Americas Corp. The Woodlands, TX). Another
dataset with better resolution collected for structure refinement was analyzed by the program XDS1.
The X-ray diffraction intensity data were converted to structure-factor amplitudes by the program
TRUNCATE of the CCP4 suite2. The crystal data and the statistics of data collections are
summarized in Supplementary Table 2. The initial phases were determined by the SAD method
using the program AutoSol of Phenix suite with figure-of-merit of 0.323-5
. Strong electron densities
of bromine atoms and silver ions were clearly observed in the initial electron density map.
Anomalous difference Fourier electron density map is shown in Supplementary Fig. 2. A molecular
model of the crystal was constructed by using the program Coot6,7
. The atomic parameters were
refined by using the program phenix.refine of the Phenix suite3,8
. The statistics of structure
refinement are summarized in Supplementary Table 2. Molecular drawings were made by using
PyMOL9. The local base-pair parameters and pseudo-rotation phase angles of sugar rings shown in
Supplementary Tables 3, 4 were calculated using the program 3DNA10,11
.
NMR spectroscopy. 1H 11-echo NMR spectra of the DNA dodecamer for the Ag-titration were
measured on a JEOL ECA 500 MHz spectrometer equipped with NM-50TH5AT/FG2 probe at 298 K.
The samples contained 100 μM single-strand DNA dodecamer (50 μM DNA duplex), 100 mM
sodium nitrate, 0-100 mM silver nitrate and 10% D2O. The solution pH was initially adjusted by
NaOH to be 7.9. Molar ratios ([Ag]/[DNA duplex]) were changed as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 24, 36, 48, 100, 200, 400, 1000 and 2000 equivalents. At each titration point at 1-12, 200, 400
and 2000 equivalents, 5.5 μl of silver nitrate solution was added. At titration points at 24-48 and
1000 equivalents, 3.3 μl of silver nitrate solution was added. In the case of 100 equivalents, 2.9 μl of
silver nitrate solution was added. In total, approximately 100 μl silver nitrate solution was added to
the initial 550 μl NMR sample solution containing the DNA duplex. The sample was denatured at
approximately 90 °C for 5 min and followed by the incubation at room temperature for the annealing
at every titration. In the data processing, exponential window function with a line-broadening factor
of 5 Hz was applied to the raw data, and followed by baseline correction using JEOL Delta software.
The spectral regions of 11.5-15 ppm and 6.5-9.2 ppm, where imino protons and base protons were
observed, respectively, are displayed at each titration point (Supplementary Fig. 3). Diffusion
constants were determined by pulsed field gradient-stimulated echo12
NMR experiments with bipolar
pulse pairs-longitudinal eddy current delay13
and excitation sculpting water suppression14
pulse
sequences. 30 1H NMR spectra, in which 3-ms z-gradient power was linearly increased from 10% to
95% (maximum: 62.3 G cm-1
), were measured on a Bruker AVANCE III HD 500 MHz spectrometer
3
equipped with a cryogenic BBO probe at 298 K. The sample condition and the annealing procedure
were similar as described above. The molar ratios ([Ag]/[DNA duplex]) were changed as 0, 2 and
100 equivalents. Diffusion time was 50 ms for 0 and 2 equivalents, and 100 ms for 100 equivalent.
Non-linear least square curve fitting was performed to determine the diffusion constants at 0, 2 and
100 equivalents by quantifying the changes of summed integration of signals from 2'/2'' and methyl
protons (Supplementary Table 5 and Fig. 4). The equation for the curve fitting15
is as follows.
where I0 is the signal intensity without gradient, D is the diffusion constant, γH is the gyromagnetic
ratio of proton, δ is the duration of gradient, Δ is the diffusion time and g is the gradient power.
Diffusion constants were obtained by the curve fitting using Igor Pro. Error bars were estimated as
95% confidence intervals of fit coefficients.
Ultraviolet melting experiment. Ultraviolet melting experiments were performed both in the
presence and absence of silver ions. Sample solutions containing 4 μM DNA dodecamer, 0-20 μM
silver nitrate, 10 mM MOPS (pH 7.0) and 100 mM sodium nitrate were prepared. The thermally
induced transitions were monitored on an ultraviolet-visible spectrophotometer (V630, JASCO).
Relative absorbance, A = (At °C – A20 °C)/(A90 °C – A20 °C), at 260 nm versus temperature for the
mixtures are shown in Supplementary Figure 5.
4
References 1. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132 (2010).
2. Collaborative Computational Project, Number 4. The CCP4 suite. Acta Crystallogr. D Biol. Crystallogr. 50, 760-763 (1994).
3. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J.,
Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R.,
Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C. & Zwart, P. H. PHENIX: a
comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213-221 (2010).
4. Grosse-Kunstleve, R. W. & Adams, P. D. Substructure search procedures for macromolecular
structures. Acta Crystallogr. D Biol. Crystallogr. 59, 1966-1973 (2003).
5. Terwilliger, T. C., Adams, P. D., Read, R. J., McCoy, A. J., Moriarty, N. W., Grosse-Kunstleve,
R. W., Afonine, P. V., Zwart, P. H. & Hung, L. W. Decision-making in structure solution using
Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582-601 (2009).
6. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2162 (2002).
7. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486-501 (2010).
8. Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W.,
Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart P. H., & Adams, P. D. Towards
automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352-367 (2012).
9. DeLano, W. L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, Palo Alto, CA,
USA. (2008).
10. Olson, W. K., Banasal, M., Burley, S. K., Dickerson, R. E., Gerstein, M., Harvey, S. C.,
Heinemann, U., Lu, X. J., Neidle, S., Shakked, Z., Sklenar, H., Suzuki, M., Tung, C. -S.,
Westhof, E., Wolberger, C. & Berman, H. M. A standard reference frame for the description of
nucleic acid base-pair geometry. J. Mol. Biol. 313, 229-237 (2001).
11. Lu, X. J. & Olson, W. K. 3DNA: a software package for the analysis, rebuilding and
visualization of three-dimensional nucleic acid structures. Nucleic Acids Res. 31, 5108-5121
(2003).
12. Tanner, J. E. Use of the stimulated echo in NMR diffusion studies. J. Chem. Phys. 52,
2523-2526 (1970).
13. Wu, D. H., Chen, A. D. & Johnson, C. S. An improved diffusion-ordered spectroscopy
experiment incorporating bipolar-gradient pulses. J. Magn. Reson. A 115, 260-264 (1995).
14. Balayssac, S., Delsuc, M. A., Gilard, V., Prigent, Y. & Malet-Martino, M. Two-dimensional
DOSY experiment with excitation sculpting water suppression for the analysis of natural and
biological media. J. Magn. Reson. 196, 78-83 (2003).
15. Stejskal, E. O. & Tanner, J. E. Spin diffusion measurements: spin echoes in the presence of a
time-dependent field gradient. J. Chem. Phys. 42, 288-292 (1965).
5
Supplementary Table 1 | Crystallization conditions
Crystal code Crystal
used for SAD phasing
Crystal
used for refinement
Temperature 293K 293K
DNA solution (1 μl)
DNA d(GGACT[Br
C]GACTCC) 2 mM 2 mM
Silver nitrate 4 mM 4 mM
Crystallization solution (1 μl)
3-Morpholinopropanesulfonic acid (pH 7.0) 50 mM 50 mM
Spermine 10 mM 10 mM
Potassium nitrate 300 mM 250 mM
2-Methyl-2,4-pentanediol 10% 10%
Reservoir solution (250 μl)
2-Methyl-2,4-pentanediol 40% 40%
Supplementary Table 2 | Crystal data, statistics of data collections
and structure refinement Crystal code Crystal used for
SAD phasing
Crystal used for
refinement
PDB-ID - 5IX7
Crystal data
Space group P6122 P6122
Unit cell (Å) a = b = 30.0, c = 118.5 a = b = 30.2, c = 118.4
Z a 1 1
Data collection
Beamline BL-5A of PF BL-1A of PF
Wavelength (Å) 0.91932 1.1
Resolution (Å) 26.0-1.6 26.1-1.4
of the outer shell (Å) 1.66-1.60 1.43-1.40
Unique reflections 7879 11942
Completeness (%) 99.9 100.0
in the outer shell (%) 100.0 100.0
Ranom b (%) 5.8 7.5
in the outer shell (%) 38.0 28.4
Redundancy 22.3 10.0
in the outer shell 22.5 9.3
Structure refinement
Resolution range (Å) 26.1-1.4
Used reflections 11939
R-factor c (%) 16.5
Rfree d (%) 18.9
Number of metal ion 5 Ag+, 2 K
+
R.m.s.d. bond length (Å) 0.019
R.m.s.d. bond angles (°) 1.3 a Number of DNA fragment in the asymmetric unit.
b Ranom = 100 Σhklj|Ihklj(+)–Ihklj()| / Σhklj[Ihklj(+) + Ihklj()].
c R-factor = 100 Σ||Fo| – |Fc|| / Σ|Fo|, where |Fo| and |Fc| are optimally scaled
observed and calculated structure factor amplitudes, respectively. d Calculated using a random set containing 10% of observations.
6
Supplementary Table 3 | Local base pair parameters of the
metallo-DNA duplex
Base pair Twist (°) Rise (Å) Propeller (°) C1'...C1' (Å)
G1-Ag-G1 -33 11.0
30 3.2
G2- Ag-C12 -28 11.1
36 2.9
C4- Ag-C11 -42 9.7
41 2.6
T5- Ag-T10 -44 9.3
42 3.3 Br
C6- Ag-C9 -40 9.4
35 2.6
G7- Ag-G7 -28 11.0
Average 37 2.9 -36 10.3
B-form 37 3.3 -11 10.7
A-form 33 2.8 -12 10.7
Supplementary Table 4 | Pseudorotation phase angles of
sugar rings in the metallo-DNA duplex Nucleotides Pseudorotation (°) Puckering
G1 161 C2’-endo
G2 161 C2’-endo
A3 131 C1’-exo
C4 9 C3’-endo
T5 170 C2’-endo Br
C6 85 O4’-endo
G7 38 C4’-exo
A8 152 C2’-endo
C9 168 C2’-endo
T10 128 C1’-exo
C11 83 O4’-endo
C12 163 C2’-endo
B-form 162 C2’-endo
A-form 18 C3’-endo
Supplementary Table 5 | Diffusion constants of the DNA
[Ag]/[DNA duplex] diffusion constant, D (10-10
m2 s
-1)
0 1.66 ± 0.05
2 1.29 ± 0.02
100 0.75 ± 0.05
7
Supplementary Figure 1 | Crystal packing (a) through AT-Ag-T triplet (b) and AA stacking (c).
Supplementary Figure 2 | Anomalous difference Fourier electron density map contoured at 4.0 σ
level. Peak height (σ) of each silver ion is indicated. For better understanding, hydrogen atoms
included in the structure refinement are not shown in this figure.
8
Supplementary Figure 3 | Ag-titration experiments with 1H NMR spectra of the DNA dodecamer.
The first column is the molar ratio ([Ag]/[DNA duplex]). The second and third columns are the 1H NMR
spectra in the imino proton and base proton regions, respectively. The signal intensity is normalized for
the respective region. The imino proton signals from T5 and T10 would be overlapped at 13.8 ppm, and
those from G2 and G7 would be separately observed around 12.5 ppm. The signal from G1 would not be
observed due to rapid proton exchange. The last column is the supposed DNA structure at each titration
point. The NMR spectra are explained with two transitions between three states. For interpretation of the
spectra, see the legend to the second page of this figure.
9
(Continued) Supplementary Figure 3 | Ag-titration experiments with 1H NMR spectra of the DNA
dodecamer. As the initial state, no imino proton signal was observed, and Ag-free DNA dodecamer may
exist as a single strand, or may form a very unstable duplex. As the molar ratio ([Ag]/[DNA duplex])
increased up to 2 equivalents, imino proton resonances emerged and increased. Similarly, in the base
proton region, original NMR signals of the Ag-free state were reduced, and new signals were emerged.
These spectral changes are consistent with the formation of a DNA duplex with two C-Ag-C base pairs
due to its molar ratio of 2. This interpretation was further supported by the following observation. From
the NMR spectra, signals of two GC base pairs were observed at 12.7 and 12.9 ppm, and one signal of the
terminal GC base pair was probably missing due to exchange with protons of bulk water molecules. For
the AT base pairs, two signals seemed to be overlapped at 14.0 ppm due to its integral value. Then, further
additions of silver ion promoted reductions of NMR signals for the DNA duplex with two C-Ag-C base
pairs, and all the imino proton signals disappeared at molar ratio ([Ag]/[DNA duplex]) of 10 equivalents.
In the case of base proton signals, sharp signals of the DNA duplex with two C-Ag-C base pairs were
gradually decreased, and were replaced with broad signals. Such broad signals strongly inspired us that
the DNA dodecamers may form oligomerized silver-DNA hybrid in solution.
10
Supplementary Figure 4 | Curve fitting of the signal decay induced by the pulsed field gradient. Red
lines indicate fitted curves. Diffusion time was 50 ms at 0 and 2 equivalents, and 100 ms at 100
equivalents.
Supplementary Figure 5 | Relative absorbance, A = (At °C – A20 °C)/(A90 °C – A20 °C), at 260 nm versus
temperature. Sample solutions contain 4 μM DNA dodecamer, 0, 4, 8, 12, 16 or 20 μM silver nitrate, 10
mM MOPS (pH 7.0) and 100 mM sodium nitrate. These melting curves suggest that the DNA dodecamer
forms a higher order structure in the presence of silver ion. In addition, the change of melting curves is
concentration-dependent up to molar ratio ([Ag]/[DNA duplex]) of 10 equivalents. These results indicate
the followings; (i) the DNA dodecamer may takes the less-stable self-complementary duplex composed of
the Watson-Crick AT and GC and water-mediated CC base pairs in the absence of silver ion, (ii) the DNA
dodecamer takes certain higher order structures in solution containing silver ion, which are likely the
silver-DNA hybrid oligomer/nanowire.
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