Polyadenosine-based fluorescent probe for reversible pH sensing based on protonated adenine-adenine base pairs: Applications to sensing of enzyme-substrate system and enzymatic logic gates

Numerous DNA-based sensors have been developed for colorimetric, fluorescent, and electrochemical detection of pH changes in an aqueous solution. However, few studies have explored the use of polyadenosine-based sensors for probing pH changes based on protonated adenine-adenine (AH+-H+A) base pairs. This study presents a fast, simple, sensitive assay for fluorescent detection of a small pH variation in an enzymatic reaction based on controlling the conformation of the polyadenosine-based probe. The DNA-based pH probe consists of a 20-mer adenosine base (A20), a Cy3 fluorophore at the 5′-end, and a BHQ2 quencher at the 3′-end. At a low DNA concentration, lowering pH activates the Cy3-A20-BHQ2 probe to fold back on itself through the formation of AH+-H+A base pairs. A short intramolecular double-stranded region is then formed in a single-stranded A20 probe. As a result, Cy3 is located close to BHQ2, leading to fluorescence quenching of Cy3 by fluorescence resonance energy transfer. The Cy3-A20-BHQ2 probe was well-suited to reversibly probe a 0.2-pH unit change in the pH range of 3.4-6. Additionally, the pH-induced conformation change of A20 allowed the Cy3-A20-BHQ2 probe to detect a slight pH fluctuation in the enzymatic reaction and construct NAND and NOR logic gates using two enzymes as inputs.