Rational design of tunable pH switches through shadow-strand hybridization-actuated displacement engineering

Abstract Local pH variations play a pivotal role in numerous critical biological processes. However, achieving the tunability and selectivity of pH detection remains a challenge. Here, we present a DNA-based strategy that enables programmable and selective pH responses, which is termed shadow-strand hybridization-actuated displacement engineering (SHADE). The tunability of pH responses is accomplished via rational manipulations of shadow strands derived from the i-motif-forming sequence. Shadow strands, which can be considered the “nucleic acid molecular chaperones,” assist in the folding of i-motif structure under acidic conditions via toehold-mediated strand displacement reactions (TMSDR). The response to alkaline conditions is achieved through a hairpin shadow (HS) containing A+-C pairs in stem. Combining i-motif-forming sequence and HS allows for the development of a narrow pH-responsive probe. Furthermore, aptamer was conjugated to guide the probe on cell surfaces. The pH sensitivity of SHADE allows for significant fluorescence enhancement in acidic tumor microenvironments, thereby improving the signal-to-noise ratio in vivo imaging. This work represents the application of TMSDR programmability to pH regulation. The SHADE strategy holds promise beyond pH sensing, potentially enabling the manipulation of diverse quadruplex architectures and facilitating the creation of highly responsive components for synthetic molecular devices and signal transduction networks.