Analysis of various sequence-specific triplexes by electron and atomic force microscopies
Analysis of various sequence-specific triplexes by electron and atomic force microscopies
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DOI：
10.1016/s0006-3495(98)74026-3
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发表时间：
1998-02-01
影响因子：
3.4
通讯作者：
Delain, E
中科院分区：
文献类型：
--
作者：
Cherny, DI;Fourcade, A;Delain, E
通过电子 (EM) 和原子力 (AFM) 显微镜观察包含 20 聚体 G、A 的三螺旋形成寡核苷酸 (TFO) 和双-PNA（肽核酸）与双链 DNA 的序列特异性相互作用。由生物素化 TFO 形成的三链体很容易通过 EM 和 AFM 检测到，其中链霉亲和素是标记物。长质粒 DNA 内未标记三联体的 AFM 图像显示，目标位点位置内双螺旋的高度增量类似于 0.4 nm。 TFO 与 74 nt 长的寡核苷酸缀合，形成 33 bp 长的发夹，与目标位点形成极其稳定的三链体，很容易通过 EM 和 AFM 将其成像为突出的 DNA。短双链体在相对于双螺旋轴的垂直方向上突出，或者在支持物的平面内或者在支持物的平面外。在后一种情况下，当三联体位点的高度增加0.3-0.4 nm时，突起的表观高度类似于1.5 nm。 bis-PNA 形成三聚体，其中 PNA 的两个十聚体通过柔性接头连接，导致目标位点双螺旋变形，这很容易被 EM 和 AFM 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，首次直接可视化了 TFO 和 PNA 与其目标序列在长质粒 DNA 内的序列特异性相互作用。
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 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 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 similar to 1.5 nm, when that of the tripler site is increased by 0.3-0.4 nm. Tripler 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 similar to 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.