Analysis of Various Sequence-Specific Triplexes by Electron and Atomic Force Microscopies

Abstract

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 approximately 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 approximately 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 approximately 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.

Time-Lapse Imaging of Conformational Changes in Supercoiled DNA by Scanning Force Microscopy

“…During the drying step, uranyl acetate is expected to confine the DNA molecules in a highly viscous layer on the mica surface, thus hindering their conformational rearrangements during the drying process. This procedure has been originally applied to SFM sample preparations by Cherny and co-workers (32) and has been applied in our study with slight modifications. The shape of molecules treated with uranyl acetate but not rinsed with water (protocol 2) showed completely different distribution of DNA topology.…”

Nanopore Based Sequence Specific Detection of Duplex DNA for Genomic Profiling

“…In accordance with Figure 3 we find that, on average, the mean translocation time of the 8-base PNA/DNA complex (∼20 μs/bp) is ∼200-fold greater than a similar length of free DNA (0.1 μs/bp). Stalling at the DNA/PNA complex site may result from a combination of two factors: a) increased interactions of the bulky, less-charged complex with the pore13, and b) formation of kinks along the DNA molecule at the PNA binding site, as observed by electron microscopy and atomic force microscopy17. Regardless of the exact stalling mechanism, this process serendipitously improves the fidelity of detecting a DNA/PNA complex in a long DNA fragment, particularly important for minimizing errors associated with multiple tag readout.…”

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

“…An alternative approach recognises that at the invasion site of a DNA/PNA complex, the conformation of the natural double-stranded DNA changes where a bulge and/or kink becomes apparent, in a highly localised and sequence-specific manner (Cherny et al, 1998). Singer and co-workers demonstrated that by using solid-state nanopores, which can be considered as single-molecule Coulter counters, it is possible to detect the PNA invasion site (Figure 9).…”