Engineering fluorinated and polarity-inverted G-quadruplex aptamers with enhanced properties and G4-ligand carrier potential

The dimeric parallel G-quadruplex (G4) aptamer INTB (T30175) possesses promising antiproliferative properties, but its therapeutic potential is limited by poor thermal stability and low nuclease resistance. To overcome these limitations, we designed and synthesized six novel fluorinated and polarity-inverted INTB analogues incorporating 3′–3′ inversion-of-polarity sites and 2′-deoxy-2′-fluoro-guanosines (GF) at strategic positions. Their physicochemical properties were characterized by circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting and isothermal competition assays, UV spectrophotometry, and NMR spectroscopy. These analyses, complemented by polyacrylamide gel electrophoresis (PAGE), confirmed that all modified analogues retain the characteristic parallel dimeric G4 architecture of the parent aptamer. Chemical modifications significantly enhanced both thermal stability and biological resistance; notably, the INTB T1 variant remained 88% intact after 72 h of incubation in serum. Biological evaluation in HCT 116p53−/− colorectal carcinoma cells identified INTB T1 and INTB D2 as the most potent cytotoxic derivatives at 5 and 10 μM. Furthermore, G-quadruplex Fluorescent Intercalator Displacement assay demonstrated that these analogues efficiently bind to small-molecule G4-ligands (360A, PhenDC3, TMPyP4) and fluorescence quenching further confirmed PhenDC3 binding. When exploited in combination with PhenDC3, the complexes showed sequence-dependent enhancement of cytotoxic activity. In particular, the INTB D2/PhenDC3 complex emerged as the most active combination, displaying greater cytotoxicity than the individual components and a response associated with caspase-dependent apoptotic signalling and altered cell-cycle regulation. Our findings highlight the potential of these engineered G4-aptamers as robust antiproliferative agents and as potential carriers of small-molecule G4-ligands.