An intra-locked RNA G-quadruplex topology confers enhanced nuclease resistance and cancer cell targeting

Nucleic acid aptamers often suffer from poor serum nuclease resistance, which limits their translational applications. Here, we present Apt-10-2, a natural 10-nt RNA aptamer that forms a highly stable G-quadruplex (Tm > 90 °C) with prolonged serum persistence (>96 h), while maintaining nanomolar affinity for nucleolin (NCL). Functionally, Apt-10-2 distinguishes cancer cells from normal cells in vitro and demonstrates specific tumor accumulation (>8 h) in vivo. To investigate the molecular basis of its stability, we performed NMR-based structural analysis and proposed a restraint-supported structural model of Apt-10-2, suggesting a unique interlocked topology in which two subunits co-assemble into a 16-layered framework composed of G-, U-, and A-tetrad-like interactions.. The proposed structural model features an intra-locked domain stabilized by cross-stacking interactions between the G1-U2-U3 tetrads, which are critical for maintaining the aptamer's stability. Disruption of this domain significantly impairs the overall structure. Our findings provide a structural framework for understanding the prolonged stability of RNA G-quadruplexes, and highlight the potential of Apt-10-2 as a naturally stable, high-affinity aptamer scaffold for cancer-targeting applications, without the need for extensive chemical modifications.