Engineering G-quadruplex aptamer-5-fluoro-2′-deoxyuridine conjugates: Impact of tail orientation and RNA spacer insertion on structure and antitumor activity

G-quadruplex-forming aptamers represent a promising class of molecules for targeted cancer therapy. However, their therapeutic potential is often limited by a low cytotoxic activity. To overcome this limitation, chemical conjugation with cytotoxic agents has been explored, although rational strategies to optimize such chimeras remain underdeveloped. To enhance the activity of the STAT3-targeting G-quadruplex aptamer (STAT), we covalently attached a 10-mer tail of 5-fluoro-2′-deoxyuridine (FdU) to either its 5′ or 3′ end of the sequence. Structural characterization revealed that the position of the FdU tail modulates the equilibrium between monomeric or dimeric species of STAT aptamer. These structural differences critically impact the chimera's biological properties, including cellular uptake, intracellular trafficking, and in vitro cytotoxicity in cancer cells. Moreover, incorporation of an RNA spacer between the aptamer and the FdU tail further increased cytotoxicity by promoting degradation and prodrug activation. Our findings demonstrate that both nature and positional orientation of a chemotherapeutic tail on G-quadruplex aptamers can be strategically employed to modulate both structural conformation and biological function. This work establishes a rational design strategy for engineering aptamer-drug conjugates with tunable structural and cytotoxic properties.