Loop topology enables programmable apoptosis in G-quadruplex aptamers

G-quadruplex (G4) aptamers are promising nucleic acid-based therapeutics, but the role of loop architecture in modulating biological activity remains poorly defined. Here, we present a controlled comparison of two rationally designed, unimolecular antiparallel G4 aptamers, 2MFT and S172, which share the same groove-width scaffold by design, while differing in loop configuration and stack number (2MFT: three stacked tetrads; S172: two stacked tetrads) but differ in loop configuration. S172 contains a central diagonal loop, while 2MFT features two diagonal loops. Both constructs exhibited similar thermal stability and low-nanomolar potency in HeLa cells; however, S172 induced more rapid and pronounced cytotoxicity at 48 h (viability: 59.0 % vs. 70.1 %), accompanied by nearly complete G₁-phase arrest and strong upregulation of pro-apoptotic genes (CASP3, BAX) and downregulation of STAT3 and BCL2. In contrast, 2MFT displayed greater selectivity for cancer cells over normal fibroblasts, indicating a broader therapeutic window. These results identify loop topology as a critical determinant of intracellular activity and support a threshold-based model of G4-mediated apoptotic activation. Our findings provide a structural framework for programmable, topology-driven aptamer design and underscore the value of integrating phenotypic, transcriptomic, and cell-cycle analyses to advance next-generation nucleic acid therapeutics.