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

Area of Science:

• Biotechnology
• Molecular Engineering
• Biophysics

Background:

• Enzyme allosteric control typically requires extensive protein engineering.
• A need exists for methods to regulate enzyme activity without altering the enzyme's structure.

Purpose of the Study:

• To introduce single-molecule DNA tweezers (SMDTs) as a novel platform for programmable, allosteric-like enzyme regulation.
• To demonstrate SMDTs' ability to control enzyme activity using user-defined chemical cues without enzyme modification.

Main Methods:

• Designed SMDTs comprising two aptamers linked by a tunable, stimuli-responsive DNA segment.
• Utilized SMDTs to non-covalently bind to enzymes, inducing an inhibitory 'pinched' conformation.
• Triggered SMDT conformational changes with specific molecular cues to release inhibition and restore enzyme function.

Main Results:

• SMDTs enabled programmable, allosteric-like enzyme regulation at nanomolar concentrations.
• Tuning DNA linker properties allowed fine control over inhibition and reactivation.
• The system demonstrated high specificity, distinguishing between similar molecular inputs, including single-base mismatches.
• Demonstrated versatility by regulating enzymes with diverse triggers: nucleic acids, transcription factors (TBP, c-Myc), signaling proteins (PDGF), small molecules (kanamycin), and metal ions (Mn2+).

Conclusions:

• SMDTs provide a generalizable framework for creating responsive protein binders.
• This platform translates molecular recognition into tunable enzyme activity control.
• Offers a novel approach to enzyme engineering and molecular sensing.