PRIORITY PAPER
Structural heterogeneity of pyrimidine/purine-biased DNA sequence
analyzed by atomic force microscopy
Mikio Kato
1,2
, Chad J. McAllister
2
, Shingo Hokabe
1
, Nobuyoshi Shimizu
3
and Yuri L. Lyubchenko
2
1
Department of Life Science, Osaka Prefecture University College of Integrated Arts and Sciences, Sakai, Japan;
2
Department of Microbiology, Arizona State University, Tempe, USA;
3
Department of Molecular Biology,
Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
We report here the direct evidence for the formation of
alternative DNA structures in a plasmid DNA, termed
pTIR10, containing a 0.23-kb pyrimidine/purine-biased
(Pyr/Pur) stretch isolated from the rat genome. Long
Pyr/Pur sequences are abundant in eukaryotic genomes, and
they may modulate the biological activity of genes and
genomes via formation of various types of triplex-related
structures. The plasmid DNA in sodium acetate buffer
(pH 4.35) was deposited on APS-modified mica, and after
drying it was imaged with an atomic force microscope in air.
Various types of thick protrusions have been observed on

strand-specific S1 nuclease, and led to the appearance of
retarded, but diffused, bands in agarose gel electrophoresis
under acidic conditions, suggesting the occurrence of
alternative DNA structure and the presence of heteroge-
neity in DNA conformations in vitro [11]. Characterization
of the potential for forming unusual DNA structure is
critical for understanding how the region works in the
genome. Traditional chemical and enzymatic probe tech-
niques were not efficient, however, in unraveling the
structural organization of such long DNA sequences as
the result was the sum of various conformers in solution.
Recently, we have successfully characterized the intramo-
lecular triplex structure formed in supercoiled DNA
within 46 bp of Pyr/Pur mirror symmetry by atomic force
microscopy (AFM) accompanied by an appropriate
sample preparation procedure [12]. Here, we apply the
same technique to visualize directly structural features of
supercoiled DNA containing long Pyr/Pur sequence
isolated from the rat genome. The studies revealed the
formation of alternative local DNA structures of different
shapes.
MATERIALS AND METHODS
DNA
A pUC19 derivative, pTIR10, containing  0.23 kb of Pyr/
Pur region within a 0.5-kb insert isolated from the rat
genome (GenBank accession number U22965 [11]) was used
in this work. Southern blot analysis revealed that the
pTIR10 sequence hybridized efficiently with fragments of
rat and human genomic DNA, meaning that similar
sequences were abundant in the genomes (M. Kato &

) was deposited on mica function-
alized with aminopropyl silatrane (APS-mica) as described
previously [13]. The AFM imaging procedure has been
described elsewhere [14]. Images were acquired by MM
SPM NanoScope III system (Veeco/Digital Instruments,
Santa Barbara, CA, USA) operating in Tapping Mode in air
Fig. 1. Electrophoretic mobility of pTIR10 DNA. Left panel, electro-
phoresis on 1% agarose in 40 m
M
Tris/acetate/5 m
M
sodium acetate/
1m
M
EDTA (pH 7.5); right panel, electrophoresis on 1% agarose in
30 m
M
sodium acetate/1 m
M
EDTA (pH 4.6). Lane 1, pUC19 DNA;
lane 2, pTIR10 DNA; lane 3, pTIR10 DNA linearized by HindIII
digestion; lane M, HindIII-digested lambda phage DNA size marker.
Fully supercoiled molecules, linear molecules, open circles and super-
coileddimermoleculesofpUC19aremarkedwitha,b,c,andd,
respectively, in the left panel. The faint DNA band at the bottom of
lanes 2 and 3 (marked with asterisk) might be the supercondensed
structure reported previously [25,26].
Fig. 2. AFM images of pTIR10 and pUC19 DNA. Structural irregu-
larities are indicated on large-scale images by arrows and the enlarged
rescanned images of the molecules are inserted. (A) pTIR10 DNA

stranded DNA is about 2 nm in B-form DNA [2]. Due to
the convolution effect of the probe tip [15,16], apparent
width of DNA obtained by the AFM will be larger than
actual size (Fig. 4D, right panel). In the present results,
differences in the parameters between the stem part and
regular DNA are close to those obtained for short
intramolecular triplexes (H-DNA) earlier [12] suggesting
that the structures observed are intramolecular triplexes.
Efficient formation of the stem structures at acidic pH also
supported the involvement of protonated bases that are
required for H-DNA. It is noticeable that heterogeneity in
size and shape of the stems occurred in the samples prepared
at acidic pH. All types of local DNA structures obtained at
acidic pH are shown in Fig. 3. An example of short stems is
shown in Fig. 3A. Relatively long stems were often curved
as shown in Fig. 3B. In the formation of intermolecular
triplex structure, nontriplex forming sequence conjugated to
the triplex forming oligonucleotide caused bending of target
DNA at the junction [17]. The curved triplex stems observed
in the present study might be caused by the presence of any
mismatch in the triplex stem. Two clearly separated stems,
twin stem structures, were also observed, and one example is
shown in Fig. 3C. The two stems can be very close and form
P-shaped and Y-shaped structures shown in Fig. 3D and E,
respectively. We have identified 45 molecules having the
triplex stems. Seven of these retained twin stems or
noncanonical triplex stems (P-andY-shaped structures)
and the rest had a single stem. Structural parameters for the
twin stems or noncanonical triplex stems (14 stems in total)
were similar to those for single stems. The average stem

molecules
Acidic sample deposition
Triplex stem 14.55 (4.28) 8.14 (0.94) 1.52 (0.25) 38
a
B-DNA
b
NA 6.03 (0.93) 0.98 (0.15) 45
Neutral sample deposition
Triplex stem 10.90 (1.43) 7.92 (1.12) 1.41 (0.33) 19
B-DNA
b
NA 6.21 (0.90) 1.03 (0.25) 19
a
The molecules having the single stem structure were used for
determining stem parameters.
b
Width and height for B-DNA were
obtained by measuring outgoing arms proximal to the stem part.
3634 M. Kato et al. (Eur. J. Biochem. 269) Ó FEBS 2002
alternative DNA structures is shown in Fig. 4. The stem
structures observed in the samples of pTIR10 DNA
prepared at neutral pH were shorter than those obtained
in the samples prepared at acidic pH, and heterogeneity in
size and shape was not seen under neutral conditions.
Long (> 200 bp) Pyr/Pur stretches are widely repre-
sented at intergenic regions in the genomes of eukaryotic
organisms. In the present work, one of the long Pyr/Pur
sequences isolated from the rat genome is shown to adopt
various types of alternative DNA structures including
multiprotrusions, supposedly involving intramolecular tri-

Japan Society for the Promotion of Science (JSPS) (to N. S.), and the
National Institute of Health grant GM 62235 (to Y. L. L.).
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Model for typical intramolecular triplex; (B) model for curved long triplex; (C) model for twin stems; (D) definition of the structural parameters.
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