ELECTRON-MICROSCOPY MAPPING OF OLIGOPURINE TRACTS IN DUPLEX DNA BY PEPTIDE NUCLEIC-ACID TARGETING
ELECTRON-MICROSCOPY MAPPING OF OLIGOPURINE TRACTS IN DUPLEX DNA BY PEPTIDE NUCLEIC-ACID TARGETING
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DOI：
10.1093/nar/22.24.5218
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发表时间：
1994-12-11
影响因子：
14.9
通讯作者：
NIELSEN, PE
中科院分区：
文献类型：
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作者：
DEMIDOV, VV;CHERNY, DI;NIELSEN, PE
生物素化同型嘧啶十聚体肽核酸 (PNA) 可与线性双链 DNA 中的互补寡嘌呤靶标形成序列特异性且稳定的复合物。使用链霉亲和素作为 EM 标记，无需化学固定，即可通过电子显微镜 (EM) 观察非共价复合物。 PNA-DNA 复合物的三重化学计量（两个 PNA 分子可能通过 Watson-Crick 和 Hoogsteen 与双链 DNA 的一条链结合）通过在一些显微照片中每个靶位点出现两个链霉亲和素“珠子”来表明，并且还通过凝胶位移测定中两个延迟带的形成来支持。对链霉亲和素“珠子”位置的定量分析表明，在优化条件下，PNA-DNA 复合物优选与完全互补的靶标形成。 PNA 浓度或孵育时间的增加会导致在含有一两个错配的位点处结合。我们的结果表明，生物素化的 PNA 可用于双链 DNA 中短靶标的电镜图谱。
Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The tripler stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson - Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.