Quantitative and sequence-specific analysis of DNA-ligand interaction by means of fluorescent intercalator probes.
A novel method of analysis of double-stranded DNA-ligand interaction is presented. The interaction is monitored by the fluorescence of a DNA bis-intercalator oxazole homodimer YoYo-3. The fluorescence intensity or its decay time reflects the modification of the DNA double helix. The DNA sequence is scanned by hybridization with short oligomers having consecutively overlapping complementary sequences to analyse the sequence specificity of binding. In our experiments we used as ligands the minor groove binders netropsin, SN6999 (both with AT-preference), the GC-specific ligand chromomycin A3 as well as the derivative SN6113 (non-specific interaction), which displace the bis-intercalator YoYo-3 or influence the duplex structure in such away that the fluorescence intensity and lifetime decrease in comparison to a ligand-free screening. The changes of fluorescence emission clearly define the binding motif and indicate minor groove interactions with a reduced DNA binding site. Titration of the ligand quantitatively characterizes its binding by determining the dependence of the binding constant on the oligonucleotide sequence.
Kirschstein
O
O
Institute of Molecular Biotechnology, Department of Single Cell and Single Molecule Techniques, Jena, Germany.
Sip
M
M
Kittler
L
L
D001665:Binding Sites; D004247:DNA / Q000032:analysis* / Q000378:metabolism*; D017931:DNA Primers; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007364:Intercalating Agents / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D009429:Netropsin / Q000378:metabolism; D009841:Oligonucleotides / Q000032:analysis / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010080:Oxazoles / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism; D011806:Quinolinium Compounds / Q000378:metabolism
D017931:DNA Primers; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D008024:Ligands; D009841:Oligonucleotides; D009842:Oligopeptides; D010080:Oxazoles; D011726:Pyridinium Compounds; D011806:Quinolinium Compounds; C412625:YoYo-3; C079604:SN 6113; D009429:Netropsin; C062476:SN 6999; D004247:DNA