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Biophys J, February 1998, p. 1015-1023, Vol. 74, No. 2
*Institute of Molecular Genetics, 123182 Moscow, Russia;
#Laboratoire de Microscopie cellulaire et
moléculaire, URA 147 du CNRS, Institut Gustave-Roussy, F-94805
Villejuif, France;
§Department of Biochemistry B, The
Panum Institute, DK-2200 Copenhagen N, Denmark;
¶Laboratoire de Biochimie-enzymologie, URA 147 du CNRS,
Institut Gustave-Roussy, F-94805 Villejuif, France; and
Department of Biochemistry, Novosibirsk Institute of
Bioorganic Chemistry, Novosibirsk 630090, Russia
Sequence-specific interactions of 20-mer G,A-containing
triple helix-forming oligonucleotides (TFOs) and bis-PNAs (peptide nucleic acids) with double-stranded DNA was visualized by electron (EM)
and atomic force (AFM) microscopies. Triplexes formed by biotinylated
TFOs are easily detected by both EM and AFM in which streptavidin is a
marker. AFM images of the unlabeled triplex within a long plasmid DNA
show a ~0.4-nm height increment of the double helix within the target
site position. TFOs conjugated to a 74-nt-long oligonucleotide forming
a 33-bp-long hairpin form extremely stable triplexes with the target
site that are readily imaged by both EM and AFM as protruding DNA. The
short duplex protrudes in a perpendicular direction relative to the
double helix axis, either in the plane of the support or out of it. In the latter case, the apparent height of the protrusion is ~1.5 nm,
when that of the triplex site is increased by 0.3-0.4 nm. Triplex
formation by bis-PNA, in which two decamers of PNA are connected via a
flexible linker, causes deformations of the double helix at the target
site, which is readily detected as kinks by both EM and AFM. Moreover,
AFM shows that these kinks are often accompanied by an increase in the
DNA apparent height of ~35%. This work shows the first direct
visualization of sequence-specific interaction of TFOs and PNAs, with
their target sequences within long plasmid DNAs, through the
measurements of the apparent height of the DNA double helix by AFM.
Biophys J, February 1998, p. 1015-1023, Vol. 74, No. 2
© 1998 by the Biophysical Society 0006-3495/98/02/1015/09 $2.00
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