Kinetic Studies by Fluorescence Resonance Energy Transfer Employing a Double-Labeled Oligonucleotide: Hybridization to the Oligonucleotide Complement and to Single-Stranded DNA

摘要: A single 16-base oligodeoxyribonucleotide was labeled at the 3'-end with fluorescein and 5'-end x-rhodamine (R*oligo*F); chromophores served as a donor/acceptor pair, respectively, for Forster resonance energy transfer. We exploited striking differences in steady-state emission spectra of R*oligo*F strand duplex structure to signal hybridization solution determine kinetics formation probe bound its oligomer complement target sequence M13mp18(+) phage DNA. The binding followed second-order kinetics; 0.18 M NaCl (pH 8) 25% formamide, rate constant 5.7 x 10(5) M-1 s-1, that 10(4) s-1. source 10-fold decrease examined differentiate between multiple nonproductive nucleation rapid fluctuations around site. From simulations based on each model combined associated experimental results, we concluded slower due structural site, an effective concentration 0.1 total. Comparisons total derived from both lifetime measurements suggest 5'-x-rhodamine induces conformational change affects interaction polymer. effects salt fluorescence were complex.(ABSTRACT TRUNCATED AT 250 WORDS)