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.
A comparative study of the stabilisation of DNA sticky ends by divalent cations was carried out by atomic force microscopy (AFM), electron microscopy and agarose gel electrophoresis. At room temperature, molecules bearing such extremities are immediately oligomerised or circularised by addition of Mg2+or Ca2+. This phenomenon, more clearly detected by AFM, requires the presence of uranyl salt, which stabilises the structures induced by Mg2+or Ca2+. DNA fragments were obtained by restriction enzymes producing sticky ends of 2 or 4 nucleotides (nt) in length with different guanine plus cytosine (GC) contents. The stability of the pairing is high when ends of 4 nt display a 100% GC-content. In that case, 95% of DNA fragments are maintained circular by the divalent cations, although 2 nt GC-sticky ends are sufficient for a stable pairing. DNA fragments with one blunt end and the other sticky appear as dimers in the presence of Mg2+. Dimerisation was analysed by varying the lengths and concentrations of DNA fragments, the base composition of the sticky ends, and also the temperature. Our observation provides a new powerful tool for construction of inverted dimers, and circularisation, ligation analysis or short bases sequence interaction studies.
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