ANALYSIS OF VARIOUS SEQUENCE-SPECIFIC TRIPLEXES BY ELECTRON AND ATOMIC-FORCE MICROSCOPIES

Sequence-specific interactions of 20-mer G,A-containing triple helix-f orming oligonucleotides (TFOs) and bis-PNAs (peptide nucleic acids) wi th double-stranded DNA was visualized by electron (EM) and atomic forc e (AFM) microscopies. Triplexes formed by biotinylated TFOs are easily detected by both EM and AFM in which streptavidin is a marker. AFM im ages of the unlabeled tripler within a long plasmid DNA show a similar to 0.4-nm height increment of the double helix within the target site position. TFOs conjugated to a 74-nt-long oligonucleotide forming a 3 3-bp-long hairpin form extremely stable triplexes with the target site that are readily imaged by both EM and AFM as protruding DNA. The sho rt duplex protrudes in a perpendicular direction relative to the doubl e helix axis, either in the plane of the support or out of it. In the latter case, the apparent height of the protrusion is similar to 1.5 n m, when that of the tripler site is increased by 0.3-0.4 nm. Tripler f ormation 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 t he DNA apparent height of similar to 35%. This work shows the first di rect visualization of sequence-specific interaction of TFOs and PNAs, with their target sequences within long plasmid DNAs, through the meas urements of the apparent height of the DNA double helix by AFM.