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NATURE CHEMISTRY | www.nature.com/naturechemistry 1

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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

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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.

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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).

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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.

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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

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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.

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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.

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(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.

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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.