Small Molecule Modulation of TERRA RNA Quadruplex Reprograms ALT-Telomere Maintenance

Telomeric repeat-containing RNA (TERRA) plays a central role in telomere maintenance in telomerase-negative cancers that employ the alternative lengthening of telomeres (ALT) pathway; however, its RNA G-quadruplex (rG4) structures remain underexplored as chemical targets. Here, we report a structure-guided approach to selectively modulate TERRA rG4 using an indoloquinoxaline-based small-molecule scaffold. Biophysical analyses confirm high-affinity and selective binding, while NMR and computational studies identify terminal tetrad stacking as the dominant binding mode of the ligand to the TERRA rG4. Ligand engagement disrupts the TERRA-Telomeric Repeat-Binding Factor 2 (TRF2) interaction (ELISA, EMSA) and alters TERRA abundance (qRT-PCR). Transcriptomic (RNA-seq) analysis reveals ligand-induced differential expression of ALT-associated genes and DNA metabolism pathways. In ALT-positive U2OS cells, the ligand localizes to the nucleus, induces telomere-associated DNA damage, and triggers S-phase arrest and apoptosis. These findings establish TERRA rG4 as a chemically addressable structural element in ALT biology and demonstrate the potential of small molecules to probe and modulate long non-coding RNA (lncRNA)-mediated genome maintenance.