Single-Molecule DNA Tweezers Enable Programmable Control of Enzyme Activity via Arbitrary Molecular Cues

Building allosteric control sites into enzymes remains a longstanding challenge, typically requiring extensive protein engineering. Here, we present single-molecule DNA tweezers (SMDTs), a unimolecular, conjugation-free platform that enables programmable and reversible allosteric-like regulation of enzymes in response to arbitrary, user-defined chemical cues. SMDTs are composed of two DNA aptamers connected by a tunable, stimuli-responsive DNA linker, which binds bivalently to distinct sites the enzyme. This dual-site binding induces a “pinched” conformation reminiscent of a mechanical tweezer and inhibits the enzyme. When desired chemical inputs are introduced, the SMDT undergoes a conformational change that dissociates the inhibitory aptamer from the enzyme, restoring its activity. The length, sequence, flexibility, and geometry of the DNA linker determine both the degree of inhibition and the efficiency of reactivation. The system operates with high specificity, discriminating between closely related inputs such as single-base mismatches in nucleic acids, and responds effectively at nanomolar concentrations. We demonstrate control of enzyme function using a range of chemical triggers, including nucleic acids, transcription factors (TBP, c-Myc), signaling proteins (PDGF), small molecules (kanamycin), and metal ions (Mn2+). These findings establish a generalizable framework for rationally designing responsive protein binders that translate molecular recognition into functional outcomes.