Rational Design of pH-Responsive Split Aptamer Probes for Controllable Ligand Binding

Modulating aptamer–ligand interactions in response to pH is essential for developing nucleic acid sensors and molecular devices capable of functioning across diverse biological pH environments. Current approaches to engineer pH-responsive aptamers predominantly rely on appending pH-sensitive motifs to the termini of the intact aptamer sequence. However, such approaches often require substantial sequence engineering, limiting their applicability to aptamers with ligand-binding pockets distant from the termini. Here, we present a rational and modular approach to pH-dependent control of aptamer function by splitting the aptamer proximal to the ligand-binding site and fusing it to a pH-responsive motif. The engineered aptamer exhibited efficient ligand binding under acidic conditions, with minimal response at neutral or alkaline pH. The findings were validated using fluorescence spectroscopy, circular dichroism (CD), electrophoretic analysis, and isothermal titration calorimetry (ITC). Notably, the engineered aptamer retained its pH-responsive behavior in a cellular environment. The design principle is general in nature and can be extended to other aptamer systems. Collectively, this work provides a versatile framework that expands the toolkit for programmable engineering of pH-responsive aptamers utilizing split aptamers, with potential applications in diagnostics and even label-free imaging using light-up aptamers.