DNA Duplex Engineering for Enantioselective Fluorescent Sensor.

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Chiral enantiomer identification is crucial for drug development and enantioselective sensor design.
• Apurinic/apyrimidinic (AP) sites exist in living cells and can be engineered into DNA.

Purpose of the Study:

• To engineer an AP site into double-stranded DNA (ds-DNA) to explore its enantioselectivity.
• To develop a straightforward chiral sensor for natural enantiomers using an AP site-specific fluorophore.

Main Methods:

• Engineered an AP site into ds-DNA.
• Utilized an AP site-specific fluorophore as an enantioselective probe.
• Employed tetrahydropalmatine (L- and D-THP) as enantiomer representatives.
• Assessed binding specificity using intrinsic fluorescence, isothermal titration calorimetry, and DNA stability.

Main Results:

• The engineered AP site selectively binds L-THP with high affinity, displacing the fluorophore and causing a significant fluorescence increase.
• L-THP showed two orders of magnitude higher affinity for the AP site compared to D-THP.
• Enantioselective performance was tunable by altering sequences near the AP site and the number of AP sites.

Conclusions:

• A single AP site in ds-DNA creates a specific binding pocket for enantioselective discrimination.
• This approach offers a novel strategy for developing efficient enantioselective sensors by engineering local DNA structures.
• The findings have implications for accelerating drug exploitation and developing advanced chiral recognition systems.