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

Abstract

Engineering allosteric control sites into enzymes typically requires extensive protein modification. Here, we introduce single-molecule DNA tweezers (SMDTs), which enable programmable, allosteric-like regulation of enzyme activity in response to user-defined chemical cues, without altering the enzyme itself. SMDTs consist of two aptamers connected by a tunable, stimuli-responsive DNA linker. By binding non-covalently to two distinct sites on an enzyme, the SMDT adopts a "pinched" conformation, reminiscent of mechanical tweezers, that inhibits enzymatic activity. Upon exposure to specific molecular triggers, the SMDT undergoes a conformational change that releases the inhibitory aptamer, restoring function. The degree of inhibition and reactivation efficiency can be finely tuned by adjusting the DNA linker's length, sequence, flexibility, and geometry. Operating at nanomolar concentrations, the system exhibits high specificity, capable of discriminating between closely related inputs, including single-base mismatches in nucleic acids. Importantly, SMDTs can be programmed to respond not only to molecular abundance but also to molecular activity. We show the versatility of this platform by regulating enzymes using diverse triggers, including nucleic acids, transcription factors (TATA-binding protein [TBP], cellular myelocytomatosis [c-Myc]), signaling proteins (platelet-derived growth factor [PDGF]), small molecules (kanamycin), and metal ions (Mn2+). These results establish a generalizable framework for designing responsive protein binders that translate molecular recognition into functional outcomes.