2-D Structure of the A Region of Xist RNA and Its Implication for PRC2 Association
In (A, B, and C), the binding sites of oligonucleotides P1 to P6 used in the FRET experiments are shown for the three 2-D structures of the A region corresponding to Models 1, 2, and 3. The identity of the chromophore present in each oligonucleotide (donor Cy3 or acceptor Cy5) is indicated in green and blue, respectively. Cy3- and Cy5-labeled oligonucleotides were purchased from Eurogentec. As illustrated in (D), the emission fluorescence spectra from 530 to 745 nm of the donor oligonucleotide bound alone to the RNA (green curve) were collected, as well as the emission spectra obtained in the presence of the donor and acceptor oligonucleotides (violin curve). No energy transfer between Cy3- and Cy5-labeled oligonucleotides was detected in solution. The FRET efficiency for each pair of oligonucleotides was defined as the decrease in fluorescence of the donor at 564 nm in the presence of the acceptor. Two representative examples of FRET assays (oligonucleotide pairs P2/P4 and P3/P6) are shown in (D). The FRET efficiencies measured for the six pairs of oligonucleotides are provided in (E) (mean values of three independent experiments). Standard deviations (σ) are shown. The relative efficiencies of the FRET obtained for each oligonucleotide pairs are schematically represented in (A, B, and C) by lines joining the oligonucleotides. The thickness of the lines reflects the efficiency of the FRET effect.