Unlocking Pb²⁺ Sensing Potential in a DNA G-Quadruplex via Loop Modification with Fluorescent Chalcone Surrogates

Area of Science:

• Biochemistry
• Molecular Biology
• Environmental Science

Background:

• Guanine (G)-rich DNA forms G-quadruplex (GQ) structures that bind toxic lead (Pb²⁺) ions, forming the basis of DNA biosensors.
• A significant challenge in Pb²⁺ detection using GQ biosensors is the competitive binding of potassium (K⁺) ions, which can interfere with accurate measurements.

Purpose of the Study:

• To develop a novel DNA biosensor strategy for highly specific and sensitive detection of Pb²⁺ ions.
• To overcome the analytical limitations posed by K⁺ ion interference in Pb²⁺ detection using G-quadruplex (GQ) based biosensors.

Main Methods:

• On-strand DNA synthesis was used to incorporate internal fluorescent chalcone surrogates into the 15-mer thrombin binding aptamer (TBA15).
• The thymidine at the 3-position (T3) of TBA15 was replaced with an indole-4-hydroxy-indanone (Ind4HI) chalcone surrogate.
• The stability of K⁺-templated GQ and Pb²⁺-templated GQ was analyzed for the modified and native TBA15 sequences.

Main Results:

• The modified T3-Ind4HI probe significantly reduced K⁺-GQ stability while increasing Pb²⁺-GQ stability.
• This modulation enhanced the specificity of Pb²⁺ binding.
• The T3-Ind4HI probe demonstrated a 15-fold increase in fluorescence intensity upon Pb²⁺ binding and could detect 6.4 nM Pb²⁺ in the presence of 10 mM K⁺.

Conclusions:

• Replacing the T3 residue in TBA15 with the Ind4HI chalcone surrogate effectively modulates metal ion affinity.
• This modification successfully addresses the analytical challenge of K⁺ interference in Pb²⁺ detection.
• The developed GQ biosensor offers a promising tool for detecting Pb²⁺ in real water samples, meeting regulatory guidelines.