A novel bioluminescent detection of exonuclease I activity based on terminal deoxynucleotidyl transferase-mediated pyrophosphate release.

Here we report a novel bioluminescence (BL) method for exonuclease I (Exo I) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated pyrophosphate release. An inert hairpin probe with a blocked protruding 3'-terminal biotinylated nucleotide was designed, and a blocked 3'-terminal nucleotide of the probe would be removed only in the presence of Exo I, thus rendering the probe with a free 3'-hydroxyl group. Subsequently, with nucleotide incorporation by TdT, huge amounts of pyrophosphates were generated. After the conversion of pyrophosphate to adenosine triphosphate (ATP) through ATP sulfurylase, BL was emitted by the reaction of d-luciferin and ATP with firefly luciferase. Therefore, Exo I activity was indirectly quantified in the range 1-500 mU with a detection limit of 0.05 mU/μl. Moreover, the developed approach was successfully applied to investigate the inhibitory effect of streptavidin on cleavage of Exo I and also determine Exo I activity in spiked serum samples. Overall, the proposed method has high sensitivity and selectivity, and can be universally extended to the detection of other nucleases using terminal extension as a signal amplification method and BL as a detection signal, having potential application in the diagnosis of nuclease-related diseases or evaluation of nuclease functions in biological systems.