A pH and Mg²⁺-Responsive Molecular Switch Based on a Stable DNA Minidumbbell Bearing 5' and 3'-Overhangs

Minidumbbell (MDB) is a non-B DNA structure of which the thermodynamic stability is sensitive to a chemical environment such as pH, serving as a potential structural motif in constructing DNA-based molecular switches. This work aims to design thermodynamically stable MDB structures bearing 5' and 3'-overhanging deoxyribonucleotides in order to examine the possibility of MDB to be functionalized. Via making use of 5-methylcytosine and adjusting the pH of solution to be acidic, MDBs bearing 1-nucleotide (nt) or 2-nt overhanging residues at the 5' and 3'-ends have been obtained. Based on one of the new MDB sequences, we have designed a molecular switch that could respond to dual inputs of pH and Mg²⁺. The MDB strand and its partner strand formed a duplex (the "ON" state) upon inputting pH 7 and Mg²⁺, whereas the duplex dissociated to restore the MDB structure (the "OFF" state) upon inputting pH 5 and EDTA. The demonstration on the ability of MDB to sustain 5' and 3'-overhanging residues and the construction of a pH and Mg²⁺-responsive molecular switch will extend the application of MDB structures in dynamic DNA nanotechnology.