Protein-free parallel triple-stranded DNA complex formation.
A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.
Shchyolkina
A K
AK
Engelhardt Institute of Molecular Biology, Russian Academy of Science, 117984 Moscow, Russia. annas@genome.eimb.relarn.ru
Timofeev
E N
EN
Lysov
Y P
YP
Florentiev
V L
VL
Jovin
T M
TM
Arndt-Jovin
D J
DJ
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000378:metabolism; D000609:Aminoacridines / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004735:Energy Transfer; D005453:Fluorescence; D005454:Fluorescence Polarization; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013816:Thermodynamics; D013941:Thymine / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics
D000609:Aminoacridines; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D051741:Kruppel-Like Transcription Factors; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D009942:Organometallic Compounds; C096797:ZNF91 protein, human; C051802:triplex DNA; C010847:9-amino-6-chloro-2-methoxyacridine; D015082:2,2'-Dipyridyl; C076491:osmium tetroxide-2,2'-bipyridine; D004247:DNA; D013941:Thymine