Characterization of RP11-350G8.5 structural features. (A) RNA secondary structure prediction of RP11-350G8.5 computed using the RNAfold web server: (left) MFE and (right) centroid structures (structures are colored by base-pairing probabilities). (B) Fluorescence emission spectra of ThT (1 μM) in the absence (black line) and presence of various RNA molecules (2.0 μM): 202, 840, 885, 1051, 1421, compared with TERRA, RG-1 positive controls, and a hairpin-forming RNA sequence (negative control). (C) Bar graph of fluorescence enhancement of ThT in the presence of the RNAs. The addition of the 5 RNA molecules resulted in fluorescence enhancements, with F_I/F_I0 values of 23, 32, 71, 81, and 20 for 202, 840, 885, 1051, and 1421, respectively. Two G4-forming positive controls, TERRA and RG-1, exhibited FI/FI₀ values of 119 and 43, respectively, whereas the negative control (hairpin) showed a value of 5. (D) Imino proton region of the 1-dimensional (1D) ¹H-NMR spectrum of 1051 recorded at 10°C. (E) Predicted secondary structures for the RNA sequences analyzed by RNAfold. (F) CD spectra of 202, 840, 885, 1051, and 1421 recorded at 10 and 100°C (solid and dashed lines, respectively). (G) ASO screening: bar graphs representative of the induction of apoptosis (fold change to vehicle) 24 and 48 hours after electroporation with 50 nM of different ASOs targeting the regions upstream (1) and downstream (2) to the 5 RNA sequences indicated as 202-840-855-1051-1421. Values on the x-axis refer to the fold change to vehicle of the percentage of cells positive for both annexin V/7AAD apoptotic markers. For the most efficient ASO (840-2), a representative annexin V/7AAD staining and a cell viability curve is provided in supplemental Figure S22B-C. Statistics were analyzed using 2-way analysis of variance test (cutoff ∗P < .05, ∗∗P < .01).