Stabilization of parallel (recombinant) triplex with propidium iodide.
Earlier we have shown that the oligonucleotide 5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-L-d(CATGCTAACT)-3' [L = pO(CH2CH2O)3p] is able to fold back forming intramolecular RecA-independent triplex with identical strands oriented parallel to each other (parallel triplex) [A.K. Shchyolkina, E.N. Timofeev, O.F. Borisova, I.A. Il'icheva, E.E. Minyat, E.V. Khomyakova, V.L. Florentiev, FEBS Letters 339, 113-118 (1994) (1)]. In this study the propidium iodide (PI) was found to intercalate into the parallel triplex and increase its stability significantly (Tm increased from 21.4 up to 44.4 degrees C in 0.01 M Na phosphate buffer, pH 7, 0.1 M NaCl, when three PI molecules per triplex were bound). Fluorescence excitation and emission spectra, the quantum yield of fluorescence (q = 0.16) and the fluorescence lifetime of PI (tau = 24.5 ns at 3 degrees C) for the parallel triplex studied were shown to be similar to those for DNA. Scatchard binding plots indicated an anticooperative mode of PI binding to the parallel triplex. The association constant is close to that of PI binding to DNA. The fluorescence experiments revealed the maximum number of binding sites to be five PI molecules per one triplex molecule. Molecular mechanics calculation of possible structures for the parallel triplex-PI complex were performed.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
O F
OF
Borisova
A K
AK
Shchyolkina
E N
EN
Timofeev
Tsybenko SYu
A D
AD
Mirzabekov
V L
VL
Florentiev
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D007364:Intercalating Agents / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D011419:Propidium / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D007364:Intercalating Agents; D009841:Oligonucleotides; D011419:Propidium; D004247:DNA