Cherny D I, Fourcade A, Svinarchuk F, Nielsen P E, Malvy C, Delain E
Institute of Molecular Genetics, Moscow, Russia.
Biophys J. 1998 Feb;74(2 Pt 1):1015-23. doi: 10.1016/S0006-3495(98)74026-3.
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.
通过电子显微镜（EM）和原子力显微镜（AFM）观察了含20聚体G、A的三链螺旋形成寡核苷酸（TFO）和双肽核酸（bis-PNA）与双链DNA的序列特异性相互作用。生物素化的TFO形成的三链体可通过EM和AFM轻松检测到，其中链霉亲和素作为标记物。长质粒DNA中未标记三链体的AFM图像显示，目标位点位置的双螺旋高度增加了约0.4纳米。与形成33碱基对发夹的74核苷酸长寡核苷酸偶联的TFO与目标位点形成极其稳定的三链体，EM和AFM都能将其作为突出的DNA轻松成像。短双链相对于双螺旋轴在垂直方向突出，要么在支撑平面内，要么在支撑平面外。在后一种情况下，当三链体位点的表观高度增加0.3 - 0.4纳米时，突出部分的表观高度约为1.5纳米。由双PNA形成的三链体，其中两个十聚体PNA通过柔性接头连接，会导致目标位点的双螺旋变形，EM和AFM都能很容易地将其检测为扭结。此外，AFM显示这些扭结通常伴随着DNA表观高度增加约35%。这项工作通过AFM测量DNA双螺旋的表观高度，首次直接观察到TFO和PNA与其在长质粒DNA中的目标序列的序列特异性相互作用。
