Stabilization of Parallel (Recombinant) Triplex With Propidium Iodide

Abstract

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) [AK. 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 (T_m increased from 21.4 up to 44.4°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 = 0.16) and the fluorescence lifetime of PI (τ= 24.5 ns at 3°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.