Harnessing Synergistic Enthalpy‐Entropy Regulation: An I‐Motif‐Based Modulating Design for Programming Stimulus‐Responsive DNA Switches

Abstract

ABSTRACT DNA switches with target‐induced allostery show great potential in biomedical application, yet often remain unprogrammable responses and limited accuracy. Existing designs also lack adaptability beyond predefined response windows and offer limited sensitivity tuning. Herein, we present an i‐motif‐based modulating design that leverages enthalpy‐entropy synergy to engineer programmable, tunable pH‐responsive DNA switches applicable to diverse aptamers. The design couples target‐binding aptamers with split i‐motif structures through variable‐length linkers that act as dual thermodynamic regulators. Linker‐length modulation permits collective control over critical parameters of switches, including target responsiveness, functional pH window, and the magnitude of pH‐dependent affinity shifts. An auxiliary sequestration mechanism further allows independent fine‐tuning of individual parameters. This design successfully converts aptamers targeting ATP, cortisol, Zn 2+ , and PTK7 into programmable switches, demonstrating versatility across different binding characteristics. Practical validation in tumor microenvironment profiling confirms the design’ capability for multi‐analyte detection with enhanced sensitivity. By establishing fundamental thermodynamic perspective and achieving cross‐platform adaptability, this strategy represents a paradigm shift from trial‐and‐error methods to rational molecular switch engineering, opening new avenues for responsive biosensing and diagnostic applications.