Unnatural nucleotide-based rkDNA probe combined with graphene oxide for detection of alkaline phosphatase activity

In this study we developed a fluorescent double-stranded DNA, incorporating an unnatural dU^rk nucleotide, that we used as a probe for the detection of alkaline phosphatase (ALP) based on enzymatic cleavage of the non-fluorescent complementary strand. Primer extension performed using the unnatural nucleotide triphosphate dU^rkTP and the natural deoxynucleotide triphosphates dATP, dCTP, and dGTP provided a simple fluorescent DNA strand that hybridized with the 5́-monophosphate non-fluorescent complementary strand. When applying the 5́-phosphate recognition and cleavage properties of lambda exonuclease (λ-exo), this probe could bind to graphene oxide (GO) and quench the fluorescence (in the absence of ALP) or not bind to GO and retain its fluorescence (in the presence of ALP). We obtained strongly fluorescent DNA strands through simple incorporation of multiple A sites in the complementary sequence, thereby increasing the number of dU^rk residues during primer extension. This unnatural nucleotide–based rkDNA probing system exhibited high fluorescence differentiation for discriminating the status of ALP. This rkDNA-GO probing system appears to be a promising tool for monitoring the activity of disease-associated enzymes.