Surface Science 539 (2003) L531–L536

www.elsevier.com/locate/susc

Surface Science Letters

Visualization of detailed structures within DNA

Hiroyuki Tanaka, Tomoji Kawai *

The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan

Received 4 January 2003; accepted for publication 3 June 2003

Abstract

Here we report ultrahigh-resolution scanning tunneling microscopy images of detailed structures within DNA

composed of a well-defined base sequence, deposited on substrates using a pulsed injection method. Poly(G–C)–

poly(G–C) and poly(A–T)–poly(A–T) have been differentiated by comparing their secondary (helical) structures. We

have visualized the structure of poly(A–T)–poly(A–T) with a resolution high enough to recognize individual nucleo-

tides. By comparing the size of individual nucleotides (the primary structure) of poly(A–T)–poly(A–T), sequencing of

individual nucleotides can be achieved successfully. These results validate the Watson and Crick DNA model at the

single molecule level 50 years after its discovery.

� 2003 Elsevier B.V. All rights reserved.

Keywords: Scanning tunneling microscopy; Surface structure, morphology, roughness, and topography; Copper; Biological mole-

cules – nucleic acids; Low index single crystal surfaces

Since discovery of the double helix structure of

DNA [1] in 1953, enormous effort has been ex-

pended to visualize detailed structures within

DNA molecules using high-resolution microscopy.

The highest resolution images were reported in1989–1990 using scanning tunneling microscopy

(STM) [2] by dropping DNA solution through air

[3–8] onto highly oriented pyrolytic graphite sub-

strates. However, later reports identified these

‘‘DNA images’’ as grain boundaries of graphite

[9,10]. After those reports, few high-resolution

STM images have appeared in scientific journals.

In order to circumvent artifacts and contamina-

* Corresponding author. Tel.: +81-6-6879-8445; fax: +81-6-

6875-2440.

E-mail address: kawai@sanken.osaka-u.ac.jp (T. Kawai).

0039-6028/03/$ - see front matter � 2003 Elsevier B.V. All rights res

doi:10.1016/S0039-6028(03)00794-5

tion, we developed a ‘‘pulsed injection method’’

[11,12] to prepare biological molecules, such as

DNA, for STM while avoiding decomposition and

aggregation. Using this method, STM produced

images of intact plasmid DNA and its periodichelical structure, but individual nucleotides of

single- or double-stranded DNA were not clearly

imaged [12,15]. Here, we present high-resolution

STM images of four well-defined types of double-

stranded DNA molecules, poly(A)–poly(T),

poly(A–T)–poly(A–T), poly(G)–poly(C), and

poly(G–C)–poly(G–C), in which a pair of single

strands forming a duplex strand and even indi-vidual nucleotides are visible. This validates the

Watson and Crick model at the single molecule

level 50 years after its discovery.

DNA cannot be thermally deposited due to

pyrolytic decomposition, therefore the standard

erved.

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method for sample preparation involves dropping

DNA-containing solution directly onto a substrate

under ambient conditions [3–8]. However, even on

well-defined and artifact-free metal surfaces, this

method results in the deposition of aggregated

DNA because of the hydrophobicity of the metal.In our experiments, a DNA solution (0.25 mg/ml)

was pulse-injected onto a well-defined Cu(1 1 1)

surface under ultrahigh vacuum using a high speed

solenoid valve (General Valve Co., Series 9)

[11,12]. Co-deposited solvent (H2O) volatized,

leaving DNA on the surface before aggregation

occurred. This pulse injection technique was de-

veloped for the deposition of biomolecules, such asDNA, while avoiding decomposition, and has

been employed successfully for a variety of mole-

cules [13,14].

Poly(A)–poly(T), poly(A–T)–poly(A–T), poly-

(G)–poly(C), and poly(G–C)–poly(G–C) (Amer-

sham Biosciences, Tokyo, Japan) were chosen for

this study because they consist of a well-defined

base pair repeating unit. According to their certi-fication, undetermined amounts of sodium chlo-

ride (NaCl) were included in their synthesis for

structural stabilization of the DNA, and these

polymers may contain a residual amount of NaCl.

They were dissolved in water made by Milli-Q

Synthesis (Millipore Co. Ltd., Tokyo, Japan) at a

concentration of 0.25 mg/ml. The sample lengths

were measured using an electrophoresis method.The mean lengths of above four samples were over

10,000 base pairs (bp), 1534, 8560 and 249 bp,

respectively.

The samples were deposited on Cu(1 1 1) sub-

strates separately using a pulse injection method.

The pulse valve, that orifice is 0.8 mm, was filled

with a DNA solution and attached to the prepa-

ration chamber in vacuum (<1� 10�7 Torr). Aclean Cu(1 1 1) substrate was located just under the

pulse valve. The distance from the pulse valve to

the substrate was 130 mm. The solution was in-

jected toward the substrate at room temperature

when the valve was opened for 1.5 ms. Amount of

injected sample solution was 0.005 ml and less.

After deposition of the DNA on Cu(1 1 1) surface,

the base pressure of the preparation chamberchanges to >1� 10�3 Torr. After the base pressureof the preparation chamber becomes better than

1� 10�7 Torr, the Cu(1 1 1) sample was transferredto the STM chamber [11,12].

Fig. 1 shows STM images and topographic

height profiles of poly(G–C)–poly(G–C) ((a) and

(b)) and poly(G)–poly(C) ((c)–(e)) obtained after

pulse injection of the sample solutions onto cleanCu(1 1 1) substrates. As shown in Fig. 1(a) and (b),

poly(G–C)–poly(G–C) molecules had a topo-

graphic height of 0.4–0.5 nm and a width (hori-

zontal diameter) of 3–4 nm. The DNA in Fig. 1(a)

appeared to be composed of bright spots separated

by approximately 3.5 nm. Since these structural

characteristics and dimensions agree well with

previous data obtained for intact plasmid DNAadsorbed on Cu(1 1 1) surfaces [12,15], poly(G–C)–

poly(G–C) molecules are duplex and each spot

corresponds to a turn of the double helix. In the

higher resolution image (Fig. 1(b)), the structure of

regularly spaced spots (the pitch of DNA) is hard

to recognize but the more detailed structures

(subnanometer particles) are visualized. At this

time we cannot completely explain the mechanismgoverning the image production. We speculate

that materials stuck on the apex of the tip play an

important role because a clean (metallic) tip tends

to facilitate the high-resolution image. The visu-

alized particles have a diameter of 0.5–0.8 nm and

appear almost randomly arranged in the DNA.

They probably correspond to a DNA component

such as base molecule, sugar-phosphate chain, andcounter ion. As shown in Fig. 1(c) and (d),

poly(G)–poly(C) molecules have a topographic

height of 0.3–0.5 nm and a width of 3–4 nm. Due

to the condition of the tip, the pitch of DNA is

difficult to recognize, but detailed structures can be

seen. Our observations revealed no appreciable

difference in the duplex characters of poly(G–C)–

poly(G–C) and poly(G)–poly(C). However, wefound poly(G)–poly(C) to be an exception. As

shown in Fig. 1(e) the strand has a topographic

height of 0.3–0.4 nm and a width of 1.2–2 nm,

which is small for duplex DNA. The strand in the

lower portion of the image is composed of oblique

bright spots aligned in a row with a separation of

approximately 0.7 nm. Considering that the single-

stranded DNA oligomers adsorbed on Cu(1 1 1)have a longer nucleotide periodicity along the

single strand of up to 0.7–0.9 nm [16], we believe

Fig. 1. STM images and topographic height profiles of poly(G–C)–poly(G–C) ((a) and (b)) and poly(G)–poly(C) ((c)–(e)) obtained

after pulse injection of the sample solutions onto clean Cu(1 1 1) substrates. Imaging parameters: (a) Vs ¼ �2 V, It ¼ 1 pA, scan of100� 53� 1 nm3 and T ¼ 91 K, (b) Vs ¼ �0:05 V, It ¼ 1 pA, scan of 40� 35� 0.8 nm3 and T ¼ 87 K, (c) Vs ¼ þ0:5 V, It ¼ 1 pA, scanof 100� 53� 1 nm3 and T ¼ 93 K, (d) Vs ¼ �0:2 V, It ¼ 1 pA, scan of 40� 15� 0.6 nm3 and T ¼ 93 K, (e) Vs ¼ �0:1 V, It ¼ 2 pA,scan of 30� 15� 0.5 nm3 and T ¼ 92 K.

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the observed oblique bright spots correspond to

nucleotides of single-stranded poly(G) or poly(C).

However, no such single-stranded structure was

observed for the poly(G–C)–poly(G–C) sample,

which may be related its higher melting tempera-

ture compared to poly(G)–poly(C) [17]. According

to Ref. [17], DNA composed of AT pairs has a

lower melting temperature than DNA composed

of GC pairs because hydrogen bonding between A

and T is weaker than that between G and C. Thus,

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poly(A)–poly(T) and poly(A–T)–poly(A–T) tend

to show single-stranded structures more frequently

than do poly(G–C)–poly(G–C) and poly(G)–

poly(C).

Fig. 2 shows STM images and topographic

height profiles of poly(A)–poly(T) ((a) and (b))and poly(A–T)–poly(A–T) ((c) and (d)) obtained

after pulse injection of the sample solutions onto

clean Cu(1 1 1) substrates. As shown in the larger

Fig. 2. STM images and topographic height profiles of poly(A)–poly(T

after pulse injection of the sample solutions onto clean Cu(1 1 1) sub

100� 84� 0.6 nm3 and T ¼ 91 K, (b) Vs ¼ �0:1 V, It ¼ 2 pA, scan of 4of an area enclosed by the dotted line in (a)), (c) Vs ¼ �4 V, It ¼ 1 pA,pA, scan of 25� 22� 0.4 nm3 and T ¼ 300 K. A contrast-magnified imarrows indicate the brighter nucleotides.

scale images (Fig. 2(a) and (c)), the DNA mole-

cules have a topographic height of 0.2–0.3 nm and

a width of 1–3 nm. These values are smaller than

those for poly(G–C)–poly(G–C) and poly(G)–

poly(C). In order to eliminate the tip artifacts

and irreproducibility that arise from samplepreparation, the sample solution containing

poly(G–C)–poly(G–C) and poly(A–T)–poly(A–T)

was deposited and imaged with STM. We can

) ((a) and (b)) and poly(A–T)–poly(A–T) ((c) and (d)) obtained

strates. Imaging parameters: (a) Vs ¼ �5 V, It ¼ 1 pA, scan of0� 22� 0.4 nm3 and T ¼ 91 K (this image is an expanded imagescan of 100� 53� 1.2 nm3 and T ¼ 88 K, (d) Vs ¼ �1:5 V, It ¼ 2age (z ¼ 0:1 nm) is superimposed in the left of the square. White

Fig. 3. STM images and topographic height profile of poly(G–

C)–poly(G–C) and poly(A–T)–poly(A–T) obtained after

pulsed-injection of a mixed sample solution of poly(G–C)–

poly(G–C) and poly(A–T)–poly(A–T) onto clean Cu(1 1 1)

substrates. Imaging parameters: Vs ¼ �2 V, It ¼ 1 pA, scan of100� 62� 1.2 nm3 and T ¼ 90 K.

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identify them unambiguously, as shown in Fig. 3,

where two types of DNA can be recognized, the

larger being poly(G–C)–poly(G–C) and the smal-

ler corresponding to poly(A–T)–poly(A–T). Thus,

differentiation of DNA molecules of different base

sequence can been achieved. In the lower section

of Fig. 2(a) and (c), the major portion of the DNA

molecule appears to be more double-stranded ra-ther than single-stranded. It is, however, obvious

that both poly(A)–poly(T) and poly(A–T)–

poly(A–T) appear less rigid compared to poly(G–

C)–poly(G–C) and poly(G)–poly(C). We believe

this originates from the weaker hydrogen bonding

between A and T. The major difference between

poly(A)–poly(T) and poly(A–T)–poly(A–T) is that

approximately one-half of poly(A)–poly(T) ap-pears to be single-stranded instead of double-

stranded, as shown in Fig. 2(b). This strand has a

topographic height of 0.2–0.3 nm and a width of

1–3 nm. Although this width is an overestimate

because of the blunt tip, oblique bright spots

aligned in a row with a separation of approxi-

mately 0.7 nm can be recognized. As discussed

previously, these oblique bright spots likely cor-

respond to nucleotides of single-stranded poly(A)

or poly(T).

We have observed the structure of poly(A–T)–

poly(A–T) with resolution capable of visualizing

individual nucleotides. In Fig. 2(d), two crossing

double-stranded DNA chains can be seen. TheDNA chain located in the center (from upper-right

to lower-left) of the image becomes a pair of single

strands consisting of bright spots aligned in a row

with a separation of approximately 0.7 nm, which

correspond to individual nucleotides. The bright-

ness (apparent height) of the nucleotides alternates,

as indicated by arrows in the contrast-magnified

image. This brightness relation also applies to thepairing rows. Since adenine has a larger atomic

skeleton and HOMO nearer to the STM bias

voltage (smaller ionization potential) compared to

thymine [18,19], we conclude that the brighter and

dimmer spots correspond to the base sequence of

dA and dT, respectively. In addition to the differ-

entiation of poly(G–C)–poly(G–C) and poly(A–

T)–poly(A–T), a pair of single strands forming adouble-stranded structure and even individual nu-

cleotides have been visualized and sequenced, val-

idating the Watson and Crick DNA model at the

single molecule level 50 years after its discovery.

Acknowledgements

We thank Drs. N. Miyoshi and M. Nishimura

for assistance in the preparation of DNA and

collection of measurements, and Drs. K. Suzuki

and M. Furukawa for helpful discussions. This

work was performed under the Center of Excel-

lence (COE) program supported by the Ministry of

Education, Science, Sports, and Culture, Japan.

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