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

ABSTRACT 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.