Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.
Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs intercalation to a single site in 3kb of DNA at the 5' triplex-duplex junction. Footprinting with diethylpyrocarbonate and dimethylsulphate revealed strong protection from modification of adenine residues within the triple-helix in concordance with their Hoogsteen pairing with the third strand, and a strong hypersensitivity to diethylpyrocarbonate at the first adenine of the duplex. This result indicates that intercalation at this site induces a conformational change at the 5' triplex-duplex junction. Furthermore, the same diethlypyrocarbonate hypersensitivity was observed with an unmodified triple-strand forming oligonucleotide and a range of intercalating molecules present in solution. Thus the 5' triplex-duplex junction is a strong binding site for some intercalating molecules and the junction undergoes a conformational change which is sensitive to diethylpyrocarbonate upon insertion of the planar aromatic chromophore. This conformational change can be used to direct a single-strand cut in duplex DNA to a defined site.
Collier
D A
DA
Laboratoire de Biophysique, Museum National d'Histoire Naturelle, Inserm U201, CNRS UA 481, Paris, France.
Mergny
J L
JL
Thuong
N T
NT
Helene
C
C
D000166:Acridines / Q000737:chemistry*; D000225:Adenine / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009710:Nucleotide Mapping; D009841:Oligonucleotides / Q000737:chemistry; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013463:Sulfuric Acid Esters / Q000737:chemistry
D000166:Acridines; D009841:Oligonucleotides; D011687:Purines; D011743:Pyrimidines; D013463:Sulfuric Acid Esters; D004247:DNA; D000225:Adenine; C007482:dimethyl sulfate; D004047:Diethyl Pyrocarbonate