Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation.
Using biotinylated oligonucleotides and streptavidin as a marker, we have visualized, with the help of electron microscopy, the triplex formation. We used the natural homopurine-homopyrimidine sequence from human papillomavirus 16 cloned within a plasmid. Under conditions favouring the formation of pyrimidine-purine-pyrimidine triplex the corresponding pyrimidine oligonucleotide formed a complex with the insert and this complex was detected by electron microscopy. Similarly, under conditions favouring the formation of pyrimidine-purine-purine triplex the corresponding purine oligonucleotide formed a stable complex detected by electron microscopy. In both cases the complexes we observed exhibited remarkable sequence specificity. Near 80% of DNA molecules carried the streptavidin marker in the correct position and very few cases of non-specific binding were detected. We conclude that the triplex mode of recognition may provide very efficient sequence-specific markers for electron microscopy of DNA.
Cherny
D I
DI
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow.
Malkov
V A
VA
Volodin
A A
AA
Frank-Kamenetskii
M D
MD
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D004247:DNA