Design of a triple-helix-specific cleaving reagent.
BACKGROUND
Double-helical DNA can be recognized sequence specifically by oligonucleotides that bind in the major groove, forming a local triple helix. Triplex-forming oligonucleotides are new tools in molecular and cellular biology and their development as gene-targeting drugs is under intensive study. Intramolecular triple-helical structures (H-DNA) are expected to play an important role in the control of gene expression. There are currently no good probes available for investigating triple-helical structures. We previously reported that a pentacyclic benzoquinoquinoxaline derivative (BQQ) can strongly stabilize triple helices.


RESULTS
We have designed and synthesized the first triple-helix-specific DNA cleaving reagent by covalently attaching BQQ to ethylenediaminetetraacetic acid (EDTA). The intercalative binding of BQQ should position EDTA in the minor groove of the triple helix. In the presence of Fe(2+) and a reducing agent, the BQQ-EDTA conjugate can selectively cleave an 80 base pair (bp) DNA fragment at the site where an oligonucleotide binds to form a local triple helix. The selectivity of the BQQ-EDTA conjugate for a triplex structure was sufficiently high to induce oligonucleotide-directed DNA cleavage at a single site on a 2718 bp plasmid DNA.


CONCLUSIONS
This new class of structure-directed DNA cleaving reagents could be useful for cleaving DNA at specific sequences in the presence of a site-specific, triple-helix-forming oligonucleotide and also for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.
Zain
R
R
Laboratoire de Biophysique, INSERM U201, Section Recherche, UMR 8646 CNRS-Muséum National d'Histoire Naturelle, UMR176 CNRS-Institut Curie, Paris, Orsay, 75231, 91405, France.
Marchand
C
C
Sun
J
J
Nguyen
C H
CH
Bisagni
E
E
Garestier
T
T
Hélène
C
C
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000187:drug effects*; D015195:Drug Design; D004492:Edetic Acid / Q000138:chemical synthesis* / Q000494:pharmacology; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D010957:Plasmids / Q000187:drug effects; D011810:Quinoxalines / Q000138:chemical synthesis* / Q000494:pharmacology
C403231:BQQ-EDTA conjugate; D009838:Oligodeoxyribonucleotides; D011810:Quinoxalines; D004247:DNA; D004492:Edetic Acid