Formation of DNA triple helices by an oligonucleotide conjugated to a fluorescent ruthenium complex.
A conjugate of a triple helix forming oligonucleotide (TFO) and the Lambda and Delta enantiomers of the ruthenium diphenanthroline dipyridophenazine complex [Ru(phen)(2)dppz](2+) was synthesized. The ruthenium complex was attached to the 5'-end of the TFO through the dppz moiety. This conjugate formed a stable triple helix with the polypurine tract (PPT) sequence from HIV proviral DNA. The thermal denaturation temperature of the triplex was increased by 12 degrees C. One remarkable property of the Delta-[Ru(phen)(2)dppz](2+) complex is a strong increase in its fluorescence when it intercalates into DNA. While the fluorescence of the oligonucleotide conjugate was very weak, the formation of a duplex with a complementary sequence or of a triple helix with a target duplex resulted in a large increase in fluorescence of the Delta enantiomer. The increase in fluorescence allowed us to follow the kinetics of duplex and triplex formation by fluorescence spectrometry. In contrast, the Lambda enantiomer gave a much smaller fluorescence change when a triplex was formed, even though the stability of the triplex was comparable to that of the Delta enantiomer. The property was ascribed to intercalation of the dipyridophenazine moiety of the Delta enantiomer into DNA and subsequent threading of the ruthenium complex through the DNA double helix. Salt effects were consistent with the involvement of DNA breathing in the formation of the intercalating complex.
Grimm
Günther N
GN
Laboratoire de Biophysique, INSERM 201-CNRS UMR 8646, Muséum National d'Histoire Naturelle, 43 rue Cuvier, 75231 Paris Cedex 05, France.
Boutorine
Alexandre S
AS
Lincoln
Per
P
Nordén
Bengt
B
Hélène
Claude
C
D004279:DNA, Viral / Q000737:chemistry* / Q000187:drug effects; D015195:Drug Design; D005456:Fluorescent Dyes; D006678:HIV / Q000737:chemistry / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D012428:Ruthenium / Q000737:chemistry*
D004279:DNA, Viral; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D012428:Ruthenium