Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.
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 triplex 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.
Demidov
V V
VV
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow.
Cherny
D I
DI
Kurakin
A V
AV
Yavnilovich
M V
MV
Malkov
V A
VA
Frank-Kamenetskii
M D
MD
Sönnichsen
S H
SH
Nielsen
P E
PE
D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000378:metabolism / Q000648:ultrastructure*; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D020135:Peptide Nucleic Acids; D011061:Poly A / Q000378:metabolism*; D019809:Streptavidin
D001426:Bacterial Proteins; D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D020135:Peptide Nucleic Acids; C051802:triplex DNA; C078138:peptide nucleic acid, T10-lysine; D011061:Poly A; D001710:Biotin; D004247:DNA; D019809:Streptavidin